The Peripheral Conversion of Thyroxine (T 4 ) into

i •
Triiodothyronine (T 3 ) and Reverse triiodothyronine (rT3)

3
 The Peripheral Conversion
of Thyroxine (T4) into Triiodothyronine (T3)
sts-
a», and Reverse triiodothyronine (rT3)

ACADEMISCH PROEFSCHRIFT

ter verkrijging van de graad van

doctor in de Geneeskunde
aan de Universiteit van Amsterdam,
?••!•
op gezag van de Rector Magnificus
dr. J.Bruyn,
hoogleraar in de Faculteit der Letteren,
in het openbaar te verdedigen in de aula der
Universiteit (tijdelijk in de Lutherse Kerk,
ingang Singel 411, hoek Spui),
op donderdag 15 november 1979 om 16.00 uur precies

door

WILLEM MAARTEN WIERSINGA

geboren te Leiden

AMSTERDAM 1979
I; Promoter : Dr. J.L. Touber
g Coreferent : Prof. dr. A. Querido
'l Copromotor : Prof. dr. M. Koster

Dit proefschrift is bewerkt op de afdeling endocrinologie (Dr. J.L. Touber)

van de kliniek voor inwendige ziekten (Prof.Dr. M. Koster en Prof.Dr. J.
Vreeken) van het Wilhelmina Gasthuis, Academisch Ziekenhuis bij de
Universiteit van Amsterdam.
Het verschijnen van dit proefschrift werd mede mogeiijk gemaakt door
steun van de Nederlandse Hartstichting en van ICI Holland B.V.
I
i*'

i: VOORWOORD
I
I Dit proefschrift is niet opgedragen aan iemand in het bijzonder, daar het
tv, verrichte onderzoek in eerste instantie mijn eigen nieuwsgierigheid heeft
1} bevredigd en ik de indruk heb dat het meer mijn eigen genoegen heeft
r' gediend dan dat van anderen. Een woord van dank aan de velen die in de
: afgelopen jaren bijgedragen hebben tot deze studies, is wel op zijn plaats,
'. daar het onderzoek zonder hun steun niet mogelijk zou zijn geweest, en
; vooral ook omdat ik met plezier terugdenk aan de onderlinge
, samenwerking.
-\ In de eerste plaats wil ik graag Dries Schellekens bedanken voor zijn
¡ betrokkenheid bij de radioimmunologische bepalingen in al deze jaren,
[ evenals Theo van Groenestein die mij ook de vereiste practische
handvaardigheid bijbracht. Toen ik kwam, had Bertie Hensgens al de basis
gelegd voor de T3-bepaling; Reinier Dalhuisen heeft later de T4-RIA
ontwikkeld. Van de vele analisten op ons laboratorium die ooit 'in de
schildklier' hebben gezeten, wil ik met name bedanken Nel Verhaest-de
Jong (die het grootste deel van de T3-bepalingen heeft uitgevoerd, en die
ook als eerste de prognostische waarde van 'haar' bepaling inzag- cf. figuur
V. 1) en Margreet Broenink (voor het in record tijd bepalen van rT3 in een
zeer grote hoevelheid monsters). Bloedmonsters krijg je niet zonder te
prikken, en Doris de Haan prikte dat het een aard had, ook al deed het haar
meer pijn dan degenen die geprikt werden. De jacht op normale personen,
van wie m.n. het jongere vrouwelijke deel tegenwoordig toch al dun
gezaaid is, is in een universiteitsziekenhuis steeds in volle gang; des temeer
waardeerde ik hun medewerking als ik hen opgespoord had. Grote steun in
het uitbreiden van de groep normalen kreeg ik van de collegae A.J.M.
Fabius ('St. Willibrord', Heiloo) en P.A. Ykelenstam ('De Buitenhof,
Amsterdam). De patiënten wil ik extra bedanken voor hun medewerking
in situaties waarin zij het toch al moeilijk hadden. De vele testen werden
uitgevoerd in de beste harmonie met Hanneke Assies. De bijzonder
hartelijke medewerking aan mijn onderzoek die ik ondervond op de
afdelingen inwendige geneeskunde (Prof. M. Koster, Prof. J. Vreeken),
infectieziekten (R.M. van der Heide) en cardiologie (Prof. D. Durrer) was
voor mij een extra stimulans. Een uitermate prettige bijkomstigheid was de
medisch-politieke scholing door Henk Lie op de cardiologie. Het
dierexperimenteel onderzoek is verricht door Ron van Noorden in een
sfeer van arcadische eendracht, die alleen verstoord werd als de ratten hem
L achtervolgden. C. Schuyt isoleerde de rattelevercellen in het Histologisch
F Laboratorium volgens een door Prof. J. James ontwikkelde techniek. Van
% de kritische op- en aanmerkingen van Prof. P. Borst, L. Offerhaus en Prof.
ï P.A. van Zwieten bij deze dierstudies, hebben wij dankbaar gebruik
l gemaakt. Veel heb ik geleerd van Herman Sallé en Ir. J. Oosting(Instituut
; voor Medische Fysica) die het grootste deel van de gegevens statistisch
f hebben bewerkt. Hun logica bleek uiteindelijk steeds sterker dan de mijne.
Van hen begreep ik dat medische tijdschriften voor statistici vaak een bron
zijn van vermaak.
Ellen Hunter wil ik hier graag hartelijk bedanken voor haar hulp bij het
typen, Chris Bor en anderen van de centrale foto- en filmdienst voor het
mooie tekenwerk en uitgeverij Rodopi voor de grote voortvarendheid
waarmee dit proefschrift werd gedrukt.
Over mijn promotor, Jan Touber, is natuurlijk veel te zeggen; het meest
heb ik wel genoten van zijn scherpe analytische geest. Prof. A. Querido en
Prof. M. Koster ben ik zeer dankbaar dat zij bereid waren het manuscript
door te lezen; hun kritische opmerkingen zijn de uiteindelijke inhoud van
het proefschrift op diverse punten ten goede gekomen.
Tot slot wil ik graag diegenen bedanken van wie ik mijn opleiding tot
internist heb ontvangen: Th.J. van Baar (Marine Hospitaal, Overveen),
Th.J. Vervaat en H.B. Schreuder (St. Lucas Ziekenhuis, Amsterdam) en
Prof. M. Koster en Prof. J. Vreeken (Wilhelmina Gasthuis, Amsterdam).

Amsterdam, september 1979

I
^ CONTENTS

H-:' Chapter I. Introduction 1

-^ • 1.1. Definition and history of the peripheral conversion of T4 1
|x 1.2. Structure and biological activity of iodothyronines 3
1.3. Production of T3 and rT3 5
1.4. Nature of the deiodination of T4 6
1.5. Brief outline of the present study 8
1.6. References 8
Chapter II. Assays 12
II. 1 Introduction 12
- References 16
11.2. Thyrotropin (TSH) 17
2.1. Ingredients 17
2.2. Assay procedure 19
2.3. Results 22 -
2.4. Discussion 26
2.5. References 29
11.3. Iodothyronines 31
3a. Reverse triiodothyronine (rT3) 34 1'
3a. 1. Ingredients
3a.2. Assay procedure
34
36
1"
3a.3. Results 40
3a.4. Discussion 45
3a.5. References 49
3b. Triiodothyronine (T 3 ) 52
3b. 1. Ingredients 52
3b .2. Assay procedure 52
3b.3. Results 53
3b.4. Discussion 54
3b.5. References 58
3c. Thyroxine (T 4 ) 60
3c. 1. Ingredients 60
3c.2. Assay procedure 60
3c.3. Results 61
3c.4. Discussion 62 .2
3c.5. References 63
H.4. Triiodothyronine uptake ratio (T 3 U) 67 •y
- References 69 ':'{

'%
V

1
 U.S. Sample handling and storage 70
Sa. Plasma versus serum samples 70
5b. Time of sampling 70
Sc. Technique of blood collection 70 t-.
Sd. Storage of plasma samples 70
5e. References 72
Chapter III Studies in euthyroid volunteers 73
111.1. Introduction 73
111.2. Subjects and methods 73
111.3. Results 75
3a. Basal values 75
3b. TRH-test 78
3c. Cord blood 84
111.4. Discussion 87
4a. Thyroid function tests: basal values 87
- distribution 87
-effect of sex 87
-effect of age 87
- effect of body weight 88
- effect of circadian and circannual rhythms 88
4b. The TRH test 90
- side effects 90
- dosages and/or routes of administration 90
- control tests 90
- the TSH response to TRH 91
- the T 3 response to TRH 93
4c. Cord blood 93
111.5. References 93
Chapter IV. Studies in patients with thyroid diseases 99
IV. 1. Introduction 99
IV.2. Patients and methods 99
IV.3. Results 99
IV.4. Discussion 109
4a. The spectrum of thyroid function disorders 109
4b. Hypothyroidism 112
-the TSH response to TRH 113
- the T 3 response to TRH 114
4c. Hyperthyroidism 114
- T3-toxicosis 114
- the TSH response to TRH 115
- the T 3 response to TRH 116
~ T4-toxicosis 117
I IV.5. References 118
Chapter V. Studies in patients with acute non-thyroidal diseases 124
V.I. Introduction 124
V.2. Patients and methods 124
V.3. Results 128
V.4. Discussion 131
— incidence of subnormal plasma T 3 131
— modulation of the peripheral conversion of T4 131
-relation between the severity of the disease and plasma T 3 133
— setpoint of the pituitary-thyroid axis 133
— role of thyroxine 134
V.5. References 136
Chapter VI. Studies in patients with acute myocardial infarction 139
VI. 1. Introduction 139
VI.2. Patients and methods 139
VI.3. Results 142
V I A Discussion 150
— reciprocal changes in plasma T 3 and rT 3 ISO
— quantitative relationship between infarct size and the
changes in the conversion of T 4 ISO
— fate of thyroxine 151
— sequence of events in thyroid hormone metabolism in
acute myocardial infarction 153
VI.5 References 153
Chapter VII. Studies on the effect of propranolol in human subjects 156
VII. 1. Introduction 156
VII.2. Patients and methods 157
VII.3. Results 160
VII.4. Discussion 163
-propranolol inhibits the 5 '-deiodination of T 4 164
- increase of T 4 and TSH during propranolol 164
- nature of the inhibitory effect of propranolol and
its biological significance 165
VH.5. References 166
Chapter VIII. Studies on the effect of propranolol in isolated rat
liver parenchymal cells 170
VlII.l. Introduction 170
VIII.2. Materials and methods 171
VIII.3. Results 172
VIII.4. Discussion 174
VIII.5. References 176 1
Chapter IX. General discussion 178
IX. 1. Physiological modulation of the peripheral conversion of
T4 178
IX.2. Pathophysiological modulation of the peripheral conver-
sion of T 4 179
IX.3. Pharmacological modulation of the peripheral conversion
ofT 4 180
IX.4. The peripheral conversion of T 4 and the pituitary-
thyroid axis: regulatory mechanisms 182
IX.5. The biological significance of the peripheral conversion
ofT 4 184
IX.6. Epilogue 186
IX.7. References 187
Summary 194
Samenvatting 197
Samenvatting voor de leek 201
Abbreviations 204
I "£'•

CHAPTER I.

i INTRODUCTION

I.I. Definition and history of the peripheral conversion of T4.

The peripheral conversion of the thyroid hormone tetraiodothyronine

or thyroxine (T4) into triiodothyronine (T3) and reverse triiodothyronine
(rT3) is defined as the extrathyroidal deiodination of thyroxine, by which
r. — depending on the site of removal of an iodine atom — either T 3 or rT3
arises. T4 was demonstrated to be present in human plasma by Taurog and
Chaikoff in 1948, and T 3 by Gross and Pitt-Rivers in 1953. Although it v/as
already shown by Albright et al in 1954 in vitro and by Pitt-Rivers et al in
1955 in vivo in man that T4 is deiodinated to T3, conversion of T 4 into T3
could not be demonstrated in human subjects in a study of Lassiter and
Stanbury in 1958. Interest in the deiodination of T 4 thus diminished, also
due to the technical problems of the measurement of T3. With the advent of
less cumbersome techniques for the measurement of T3 in the late 1960's,

Figure I.I.
Sequential deiodination of T4, according to Chopra et al (1978). Outer ring deiodination is
indicated by open circles, inner ring deiodination by solid circles. Dashed lines indicate
proposed deiodination pathways which have not yet been demonstrated conclusively.
 and especially since the introduction of the T 3 radioimmunoassay in the
early 1970's, the fate of T 3 in health and disease was studied extensively.
Braverman et al (1970) demonstrated conclusively that T3 was generated
from T4 outside the thyroid in athyreotic human subjects, in whom
substitution therapy with T 4 was the only possible source of T3. In the same
year conversion of T4 to T, was also found in normal human subjects
(Sterling et al 1970). Many other reports have appeared, all indicating a
substantial extrathyroidal production of T3 from T4 (Pittman et al 1971;
Nicoloff et al 1972; Braverman et al 1973; Surks et al 1973; Inada et al 1975;
Nomura et al 1975). Reverse T3 (rT3) was first shown to be present in
human plasma by Chopra (1974), and the extrathyroidal production of rT3

NH 2
CH 2 -CH
COOH
PHENOLIC j/3- RING ETHER j tt-RING ALANYL
HYDROXYL |OUTER RING BRIDGE INNER RING SIDE CHAIN
GROUP I PHENOLIC RING iTYROSYL RING]
I

thyronine position

3 5 3' 5'
T4 I I I
T3 I I H
rT
3, H I I
3,3'-T2 H I H
I H H
3,5- H H I I
3-Tt H H I H
I
3-T»
To H
H
H
H
H
H
H
1
l\
Figure 1.2.
The constitution of the thyronine molecule and its iodinated derivatives (I is iodine, H is
hydrogen).
 in man has been demonstrated convincingly (Chopra 1976; Gavin et al
1977; Smallridge et al 1978; Suda et al 1978). The deiodination of T4 to T 3
V.- and rT3 appears to be the first step in a cascade of sequential deiodinations
of T4, which ultimately leads to the formation of T o via several forms of
triiodinated, diiodinated and monoiodinated thyronines as illustrated in
figure I.I (Chopra et al 1978; Sakurada et al 1978; Rudolph et al 1978;
Gavin et al 1978; Smallridge et al 1979; Geola et al 1979). The
iodothyronines 3,5-T2, 3-3'-T2, 3',5'-T2 and 3-T, are already known to
exist in human plasma (Chopra et al 1978; Burman et al 1978; Meinhold
and Schiirnbrand 1978; Smallridge et al 1979; Faber et al 1979).

1.2. Structure and biological activity of iodothyronines.

The structure of the various iodothyronines (or according to Cody

(1978) more precisely the "constitution", which refers to the connectivity of
the molecule) is presented in figure 1.2. Triiodothyronine (or 3,S,3'-T3) has
two iodine atoms in the inner ring and one in the outer ring; just the reverse
is true for 3,3',5'-triiodothyronine of rT3, hence the adjective 'reverse'.
The various iodothyronines differ greatly in biological activity. The
metabolic activity by weight of T 3 is three to four times greater than that of
T4 as tested in the bioassay, and is virtually absent in rT3 (Money et al 1960;
Pittman and Barker 1962; Barker et al 1965). It is natural to explain this
difference from structure-activity relationships. The molecular constitu-
tion and conformation (according to Cody (1978) referring to the total
geometric distribution or disposition of the atoms in three dimensions) of
T4, T3 and rT3 are depicted in figure 1.3. The structural requirements for
thyroid hormone activity are the presence of nonpolar groups (halogen or
methyl) on the 3 and S positions, and the minimal energy conformation of
mutually perpendicular planes of the phenyl rings and the 120° angle
between the rings; the activity of this primary structure is enhanced by a
halogen atom in the 3' position, but a second substituent in the 5' position
reduces activity (Jorgensen 1978). Thus, removing one inner-ring iodine
atom (as is the case in rT3) results in greatly diminished activity. Indeed,
Koerner et al (1975) found that the binding to nuclear receptor sites is
moderately strong for T4 and very weak for rT3 in comparison to T3. This
does not mean that rT3 is without biological effect. In fact, rT3 has been
shown to be more potent than T 3 in some in vitro studies: it stimulates the
erythropoietin-facilitated erythropoiesis (Golde et al 1977), the activity of
L-T3 aminotransferase (Fishman et al 1977), the amino acid uptake by
thymocytes (Goldfme et al 1976) and the growth hormone secretion by
GH, cells (Papavasiliou et al 1977). An anti-thyroxine and antimetabolic
effect of rT3 has also been demonstrated (Pittman and Barker 1959;
Pittman et al I960). However, in all these studies pharmacological doses of
rT3 were used, and the physiological significance of these actions is
therefore questionable.
 &
£

I '
HO CH, - C H
*• I
COOH

NH,

NH,
HO °\ VCH2-CH
COOH
I

Figure 1.3.
The molecular constitution (left panels) and conformation (middle and right panels) of T4, T3
and rTj. (The support given by Dr. R. Dalhuisen in the construction of these models is grate-
fully acknowledged).
 U. Production of T3 and rT3.

The production of T3 and rT3 occurs in the thyroid gland and

extrathyroidally. Precise estimation of the relative contributions of the
thyroid gland and of the peripheral tissues to the daily production of T 3
and rT3 is difficult because published data on the plasma concentrations,
the production rates (PR) and the metabolic clearance rates (MCR) differ.
Nevertheless, some conclusions can be drawn from these kinetic studies
tl; (figure 1.4). As calculated from the plasma concentration and the MCR the
daily production of T3 is 34-72 nmol (representing the lowest and highest
values), that of rT3 is 23-100 nmol. If it is assumed that the thyroidal
secretion of T3 and rT3 is related to the proportion in which they are
present in thyroglobulin (Inoue et al 1967; Abrams and Larsen 1973), the
daily thyro-ial secretion of T3 (and rT3) can then be assessed from the
product of the T 3 /T 4 (and rT3/T4) ratio in thyroglobulin and the daily
secretion of T4. As far as presently known, the thyroid gland is the only
source of T4. The daily production ( - secretion) of T4 is appr. 116 nmol,

T3/T4= 0.051-0.096 rT3/T4= 0.013-0.047

/ \
5.9-11.1 nmol T3/day 1.5-5.5 nmol rT3/day
(8.2-32.7% PR - T3) (1.5-23.7* PR-rT3)

/
\ r
\
PR-T3 PR-Ta- PR-rT3
22-66 nmol T3/day 17-98 nmol rT3/day
34-72 -vll6 23-100
nmoi/day (65-92* PR-T3) nmol/day (74-98% PR-rT3) nmol/day

SERUM T 3 1.69-2.77 nmol/1 SERUM rT 3 0.28-0.92 nmol/1

HCR 20-26 l/70kg/day MCR 82-108 l/70kg/day

Figure 1.4.
Quantitation of the relative contributions of the thyroid gland and of the peripheral tissues to
the daily production of T3 and rT3 (according to Chopra et al (1978) withminor modifications;
PR=production rate, MCR=metabolic clearance rate).
8 33% 46%
38 nmol/day 56 nmol/Uay

CH 2 -CH

(COOH)
r

CONJUGATION
I
DEAMINATION
(TIO
(glucuronide, sulphate) DECARBOXYLATION
DXY

| NON-DEIODINATION
I
RCTHWAYS: 19%. 22 nmol/day

7.5.
The molecular constitution of thyroxine with an estimate of its various routes of metabolism
(daily T4 production -"116 nmol).

and the thyroidal secretion of T3 is calculated as 5.9-11.1 nmol/day, and

that of rT3 as 1.S-S.5 nmol/day. Consequently, the peripheral conversion
of T4 generates 22-66 nmol T3 and 17-98 nmol rT3 per day. It can therefore
be concluded that the bulk of the daily production of T 3 and rT3 is
generated extrathyroidally, and that thyroidal secretion of T3 and
especially of rT3 contributes relatively minor amounts to their daily
production. It thus appears that deiodination is a major mechanism in the
degradation of T4; about 80-90% of T 4 is converted to T3 and rT3 (Chopra
1976; Gavin et al 1977). Other routes in the metabolism of T 4 (figure 1.5)
are excretion in urine and bile as T4 and T4 conjugates accounting for appr.
15% of the daily PR of T4 (Oddie et al 1964), and deamination and
decarboxylation accounting for appr. 4% (Chopra et al 1978).

1.4. Nature of the deiodination of T4.

The conversion of T4 into T3 (and rT3) in vitro has now been

demonstrated in several human tissues — in fibroblasts (Refetoff et al
1972), in liver and kidney cells (Sterling et al 1973), in lymphocytes (Holm
et al 1975) and in polymorphonuclear leucocytes (Woeber 1978) — and in a
wide variety of animal tissues (Albright and Larsen 1954; Rabinowitz and
Hercker 1971; Hesch et al 1975; Visser et al 1975; Chopra 1977;
Chiraseveenuprapund et al 1978). In fact, the in vitro conversion of
exogenously added T 4 by tissues has been used as a model in the study of
the nature of the deiodination of T 4 and the physiological, pathological

iD
f$ and pharmacological factors involved. Considerable differences in the
|T capability to deiodinate T 4 between various tissues have been found, the
W liver and kidneys demonstrating the greatest deiodination capacity
fv (Chopra 1977). This does not mean that other tissues contribute little to the
j?" extrathyroidal production of T3 and rT3, since e.g. muscles by their great
I mass might be an important source and may quantitatively equal the
y kidneys and liver in this respect. It may be postulated that all thyroid
V: hormone responsive tissues have the capacity for the deiodination of T4.
,: The deiodination of T 4 seems to be enzymic in nature since the in vitro
process is dependent on temperature, pH and substrate concentration
(Visser etal 1975; Chopra 1977; Hiifneretal 1977; Kaplan and Utiger 1978;
Chiraseveenuprapund et al 1978; Hoffken et al 1978; Woeber 1978;
Chopra et al 1978; Hiifner and Grussendorf 1978; Visser et al 1978). i
However, so far all attempts to isolate the deiodinating enzymes in pure
form have failed. The enzymic activity for the outer ring deiodination has
been located in the plasma membranes of kidney cells (Chiraseveen-
uprapund et al 1978; Leonard and Rosenberg 1978) and in the ;
endoplasmatic reticulum of liver cells (Visser et al 1976; Maciel et al 1979;
Fekkes et al 1979; Kdhrle and Hesch 1979). Evidence is accumulating that •
the complete cascade of deiodination reactions of T4 is governed by an
outer ring or 5'-deiodinase and by an inner ring or 5-deiodinase, and that
these two enzymes are not the same. The existence of two different
enzymes is further suggested by clinical data: after birth plasma T3
increases within hours to values comparable to those found in adults
whereas rT3 remains elevated for several days (Chopra et al 1975), and
prolonged reduction of dietary intake is associated with a transient
increase in rT3 whereas T3 remains low (Visser et al 1978).
The presence of sulfhydryl groups is essential for the deiodination of T4 ;
to T3 and of rT3 to 3,3'-T2 (Visser et al 1976; Chopra 1978; ]
Chiraseveenuprapund et al 1978; Visser et al 1978; Chopra et al 1978; \
Woeber 1978). The deiodinating activity is stimulated by reduced \
:
glutathione and several thiol agents (dithiothreitol, mercaptoethanol), and
it is inhibited by oxidized glutathione, sulfhydryl-oxidizing agents
(diamide) and sulfhydryl-binding agents (N-ethylmaleimide, mercuric
chloride). The sulfhydryl groups acts as cofactor, and recently Visser et al r
(1979) have proposed the following 'ping-pong mechanism' for the
;| deiodination of T4: •

1
•j T4 + E-SH 5=t T 4 . E-SH —r*E-SI
-» » E-SH + HI
i
f
\ \ A I
i. T3 2SH S-S $
:
'i
I where E-SI is the intermediate sulphenyl iodide of the enzyme, which is 'j
I reduced by the cofactor. '{
 8

Other regulatory mechanisms in the deiodination proces have been

proposed, such as changes in temperature and pH (Hflffken et al 1978),
substrate-induction of the deiodinating enzymes (Grussendorf and Hiiffner
1977) and the concentration of iodothyronines themselves. It is highly
questionable, however, whether all these factors are operative in vivo.
Although it has been shown in vitro that the deiodination reactions are
dependent on temperature and differ in their optimal pH (varying between
6.5 and 9.5), such wide changes are highly unlikely to occur in vivo. Also,
the physiological significance of the depressive action of rT3on the T 4 -T 3
conversion and of T 4 on the rT3*3,3'-T2 conversion as demonstrated in
vitro (Chopra 1977; Kaplan and Utiger 1978; Hoffken et al 1978) remains
questionable.

1.5. Brief outline of the present study.

The aim of this study is to delineate several physiological, pathological

and pharmacological factors involved in the peripheral conversion of T4.
The assays used are described in chapter II.
The determination of normal values of these tests under basal
circumstances and after stimulation with thyrotropin-releasing-hormone
(TRH) is presented in chapter III, and some physiological factors which
may modulate the conversion of T4 are discussed.
The results of the thyroid function tests in patients with thyroid disease are
presented in chapter IV. The test results are interpreted both in terms of
their value in the diagnosis of disordered thyroid function and of altered
conversion of T4.
The peripheral conversion of T4 was further evaluated in patients with
acute non-thyroidal diseases. The incidence and cause of subnormal
plasma T3 concentrations in such patients was investigated, together with
its effect on the pituitary-thyroid axis. A relationship (both qualitatively
and quantitatively) was sought between the changes in the conversion of T4
and the severity of the disease (chapters V and VI).
The effect of propranolol on the deiodination of T4 in human subjects and
in animals is described in the chapters VII and VIII.

1.6. References.

Abrahams GM, Larsen PR. Triiodothyronine and thyroxine in the serum and thyroid glands
of iodine deficient rats. J Clin Invest 1973; 52:2S22-31.
Albright EC, Larson FC, Tust RH. In vitro conversion of thyroxine to triiodothyronine by
kidney slices. Proc Soc Exp Biol Med 1954} 86: 137-40.
Barker SB, Shimada M, Makiuchi M. Metabolic and cardiac responses to thyroxine analogs.
Endocrinology 1965; 76: 115-21.
BravermanLE, IngbarSH, Sterling K. Conversion of thyroxine (T4) to triiodothyronine
(Tj) in athyreotic human subjects. J Clin Invest 1970; 49: 855-64.
 Braverman LE, Vagenakis A, Downs P, Foster AF, Sterling F, IngbarSH. Effects of
replacement doses of sodium-L-thyroxine on the peripheral metabolism of thyroxine and
triiodothyronine in man. J Clin Invest 1973; 52: 1010-7.
* v: Burman KD, Wright FD, Smallridge RC, Green BJ, Georges LP, Wartofsky L. A radio-
¡mmunoassay for 3',5'-diiodothyronine. J Clin Endocrinol Metab 1978; 47: 1059-64.
Chiraseveenuprapund P, Buergi U, Goswami A, Rosenberg JN. Conversion of L-thyroxine
to triiodothyronine in rat kidney homogenate. Endocrinology 1978; 102: 612-22.
Chopra IJ, A radioimmunoassay for measurement of 3,3',5'-triiodothyronine (reverse T3).
J Clin Invest 1974; 54: 583-92.
Chopra IJ, Sack J, Fisher DA. Circulating 3,3',5'-triiodothyronine (reverse T3) in the human
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3,3',5'-triiodothyronine (reverse T,) in man. J Clin Invest 1976; 58: 32-40.
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1099-1106.
Chopra LI, Geola F, Solomon DH, Maciel RMB. 3',5-Diiodothyronine in health and disease:
studies by a radioimmunoassay. J Clin Endocrinol Metab 1978; 47: 1198-1207.
Chopra IJ, Solomon DH, Chopra U, Wu SY. Fisher DA, Nakamura Y. Pathways of meta-
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Chopra IJ. Sulfhydryl groups and the monodeiodination of thyroxine to triiodothyronine.
Science 1978; 199: 904-6.
Cody V. Thyroid hormones: crystal structure, molecular conformation, binding, and struc-
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metabolism. J Clin Endocrinol Metab 1979; 48: 611-7.
Fekkes D, Overmeeren E van, Visser TJ. Endoplasmic reticulum location of rat liver
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d' Endocrinol 1979; 40: 22A.
Fishman N, Huang YP, Tergis DC, Rivlin RS. Relation of triiodothyronine and reverse
triiodothyronine administration in rats to hepatic L-triiodothyronine aminotransferase
activity. Endocrinology 1977; 100: 1055-9.
Gavin L, Castle J, McMahon F, Martin P, Hammond M, Cavalieri RR. Extrathyroidal con-
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(Tj) in humans. J Clin Endocrinol Metab 1977; 44: 733-42.
Gavin LA, Hammond ME, Caste JN. Cavalieri RR. 3,3-Diiodothyronine production, a
major pathway of peripheral iodothyronine metabolism in man. J Clin Invest 1978; 61:
1276-85.
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of 3',5'-diiodothyronine and 3,3'-diiodothyronine in man. J Clin Endocrinol Metab 1979;
48: 297-301.
Golde DW, Bersch N, Chopra I J, Cline MJ. Thyroid hormones stimulate erythropoiesis in
vitro. Brit J Haematol 1977; 37: 173-7.
I') Goldfine ID, Smith GJ, Simons CG, Ingbar SH, Jorgensen EC. Activities of thyroid hor-
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I 1952; 1: 439-41.
Grussendorf M, Hflfner M. Induction of the thyroxine (T4) to triiodothyronine (T3) con-
 10
verting enzyme in rat liver by thyroid hormones and analogs. Clin Chim Acta 1977; 80:
61-6.
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system in rat liver homogenate. Clin Chim Acta 1977; 78: 251-9.
Hilfner M, Grussendorf M. Investigations on the deiodination of thyroxine (T4) to 3,3'-
diiodothyronine (3,3'-T2) in rat liver homogenate. Clin Chim Acta 1978; 85: 243-51.
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triiodothyronine outer ring monodeiodinating activities. Endocrinology 1979; 104:365-
71.
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1959; 197: 1271-4.
f
1
if
 11
Pittman CS, Barker SB. The calorigenic effects of some thyroxine analogs on intact animals
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If Pittman CS, Chambers Jr JB, Read VH. The extrathyroidal conversion rate of thyroxine into
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t
Pittman JA, Tingley JO, Nickerson JF, Hill Jr SR. Antimetabolic activity of 3,3',5'-triiodo-
DL-thyronine in man. Metabolism 1960; 9: 293-5.
Rabinowitz JL, Hercker ES. Thyroxine: conversion to truodothyronine by isolated perfused
rat heart. Science 1971; 173: 1242-3.
Refetoff S, Matalon R, Biaggi M. Metabolism of L-thyroxine (T4) and L-triiodothyronine
(T3) by human fibroblasts in tissue culture: evidence for cellular binding proteins and
conversion of T4 to T3. Endocrinology 1972; 91: 934-47.
Rudolph M, Sakurada T, Fang S-L, Vaganakis AG, Braverman LE, Ingbar SH. Appearance
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46: 923-8.
Sakurada T, Rudolph M, Fang S-L, Vaganakis AG, Braverman LE, Ingbar SH. Evidence
that triiodothyronine and reverse triiodothyronine are sequentially deiodinated in man.
J Clin Endocrinol Metab 1978; 46: 916-22.
Smallridge RC, Wartofsky L, Desjardins RE, Burman KD. Metabolic clearance and produc-
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subjects. J Clin Endocrinol Metab 1978; 47: 345-9.
Smallridge RC, Wartofsky L, Green BJ, Miller FC, Burman KD. 3'-L-Monoiodothyronine:
development of a radioimmunoassay and demonstration of in vivo conversion from
3',5'-diiodothyronine. J Clin Endocrinol Metab 1979; 48: 32-6.
Sterling K, Brenner MA, Newman ES. Conversion of thyroxine to triiodothyronine in
normal human subjects. Science 1970; 169: 1099-1100.
Sterling K, Brenner MA, Saldanha VF. Conversion of thyroxine to triiodothyronine by
cultured human cells. Science 1973; 179: 1000-1.
Suda AK, Pittman CS, Shitnizu T, Chambers Jr JB. The production and metabolism of
3,5,3'-triiodothyronine and 3,3',5'-triiodothyronine in normal and fasting subjects. J Clin
Endocrinol Metab 1978; 47: 1311-9.
Surks MJ, Schadlow AR, Stock JM, Oppenheimer JH. Determination of iodothyronine
absorption and conversion of L-thyroxine (T4) to L-triiodothyronine (T3) using turnover
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Taurog A, Chaikoff IL. On the nature of the circulating thyroid hormone.J Biol Chem 1948;
176: 639-56.
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Visser TJ, Does-Tobe I vd, Docter R, Hennemann G. Subcellular localization of a rat liver
enzyme converting thyroxine into tri-iodothyronine and possible involvement of essential
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I concentrations during prolonged reduction of dietary intake. Metabolism 1978; 27:405-9.

Visser TJ, Fekkes D, Docter R, Hennemann G. Sequential deiodination of thyroxine in rat
liver homogenate. Biochem J 1978; 174: 221-9.
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rat liver enzymes catalysing the reductive deiodination of iodothyronines. J Endocrinol
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Woeber KA, L-Triiodothyronine and L-reverse-triiodothyronine generation in the human
polymorphonuclear leucocyte. J Clin Invest 1978; 62: 577-84.
I
 I
£••'
CHAPTER II

ASSAYS
I,
//./. Introduction.

The laboratory tests used in this study are competitive protein-binding

assays, mainly radioimmunoassays. The competitive inhibition principle
of radioimmunoassay is presented by the following competing reactions
(Yalow and Berson 1971):

labeled antigen specific antibody labeled antigen-

antibody complex
Ag* Ab An* Ah
(F)

Ag unlabeled antigen
in known standard

I!
solutions or
unknown samples

AgAb
unlabeled antigen-
antibody complex

Unlabeled hormone competes with labeled hormone for specific binding

sites on the antibody, thereby inhibiting the binding of labeled hormone.
Consequently, as the concentration of the unlabeled hormone increases,
the ratio of antibody-bound (B) to free (F) labeled hormone will diminish.
The concentration of hormone in an unknown sample is obtained by
comparing the inhibition observed with that produced by standard
solutions containing known amounts of hormone ("standard hormone").
This is conveniently done by plotting the B/F ratio (or the percentage
bound B) versus the hormone concentration in the standard solutions.
From the obtained "standard curve" one can find the hormone
concentration that corresponds to the B/F ratio observed in the unknown
sample.

ss.
 13

I;
I
I:

hormone concentration

Figure ILL
Standard curve and equations, giving definition of precision and sensitivity, i.e. the
detection limit (modified according to Ekins 1974).
AB O = error in B Q ABX = error in B x
AX O = error in B (when x=o) AX = error in X
AB 0
= ,the -,the
slope at B x
slope at B o
precision of X
detection limit

Since the purpose of the radioimmunoassay method is to measure

unknown hormone concentrations accurately, the method must be both
precise and not influenced by non-specific factors (Ekins 1974).
The concept of precision is illustrated in figure II. 1. The precision of the
measurement of a concentration of hormone X is dependent both on the
slope of the standard curve and on the error in the measurement of the
response metameter (i.e., the percentage bound B) at the corresponding
point. The magnitude of the error in measuring B can be calculated as the
difference of x + 2 . SD and x - 2. SD for a number of measurements of B
(AB).When X is zero, the corresponding value of AX(i.e.,A B0)defines the
detection limit or "sensitivity" of the system. Since both the slope and AB
change at different points on the standard curve, AX will vary as a function 1
of X. The intra-assay coefficient of variation($Bx!00%) can serve as an 3
indicator for the precision of the measurements on each point of the
standard curve.The error AB is the net result of two independent errors, i.e.
the "experimental" errors arising from pipetting and all other
manipulations during the assay, and the counting errors inherent in
 14
radioactivity measurements. The choice of the many variables in each
assay (e.g. concentration of reagents, times of incubation and counting)
may be facilitated by mathematical approximation of the optimal
conditions for reacting equilibrium between antigen and antibody binding
sites. We have determined the optimal conditions by trial and error.

Since radioimmunoassays compare an unknown concentration of the

hormone in the system with known concentrations of the same hormone in
a set of standards, the incubation mixtures should theoretically be
identical in every respect with the exception of the concentration of the
hormone to be measured. Addition of plasma without the hormone under
test to the standard tubes is a first step in meeting this requirement, but
complete identity between the unknown and standard incubation mixtures
is impossible to achieve in practice, and hence non-specific effects may be
encountered. Due to the immunological nature of the reactions, cross-
reactions may occur with closely related antigens and with hormonal
fragments. Due to the chemical nature of the reactions the result may be
influenced by the composition of the medium, by the presence of other
substances reacting with antigen or antibody, and by the separation of
bound and free hormone. The non-specific effects in radioimmunoassay of
hormones may thus be due to (Berson and Yalow 1973) hormonal cross-
reactivity and non-hormonal effects (pH, ionic environment, anti-
coagulant, temperature, variable damage of labeled hormone).
The labeled hormone molecule may disrupt by decay of a radioiodine
atom. Labeled fragments or free radioiodine are produced, and the
radioactivity is no longer associated only with unaltered molecules. This
phenomenon of decay catastrophe (Berson and Yalow 1973) shortens the
shelf life of the labeled hormone and diminishes immunoreactivity of the
tracer.
Peptide hormones are subject to proteolytic damage by enzymes in
blood. Damage to the labeled hormone is therefore in general greater in
plasma (particularly when hemolysed) than in buffer solution. This effect
can be minimized by using a low plasma concentration in the incubation
mixture. Since plasma vary in their proteolytic effects on hormones,
control incubations must be performed. Control mixtures or "blanks"
contain no antiserum but are otherwise identical to the incubation
mixtures of the plasma sample to be assayed, and are treated in the same
manner. Controls of the standard curve are also performed. If for example
93% of the labeled hormone in the control tube separates with the free
fraction at the time of starting the assay, the remaining 7% has thus
aspecifically been bound, due to damage to the label and to incomplete
separation of bound and free hormone fraction. If at the end of the
incubation 87% separates with the free fraction, another 6% of the labeled
hormone is aspecifically bound due to incubation damage. If
approximately the same results are obtained in the controls for standards
 15
and for unknown samples, no correction may be needed for incubation
damage and/or lack of complete separation. Assays in which the damage
to labeled hormone exceeds 10% probably should be discarded.
Uncorrected damage of less than 10% to labeled hormone however can
result in great errors in the measurements of unknown samples (Berson
and Yalow 1973).

Furthermore, tests for qualitative and quantitative specificity must be

carried out. First, similarity, or rather the absence of clear nonlinearity
must be demonstrated between the standard curve and a dilution curve of
I
an unknown sample, i.e. the hormone concentration in the unknown
sample must be a linear function of the dilution factor (parallelism).
Although parallelism is a condition which has to be met, it is in itself not
sufficient to establish immunochemical identity between the standard
hormone and the hormone in the unknown sample. Second, exogenous
hormone added to the incubation mixture, should be recovered
quantitatively in the assay (recovery).

The results of hormone determinations by radioimmunoassay methods

must be subject to quality control (Jeffcoate 1978). In our laboratory each
sample is assayed in duplicate or triplicate, the result being rejected when
their coefficient of variation is greater than the mean coefficient of
variation + 2 SD of plasma with a corresponding hormone concentration,
as determined in previous experiments. A drift within an assay is detected
by putting three samples of the same plasma at different points in one assay
run; the position of the tubes in the assay should not influence the
outcome. Reproducibility is tested by including three known plasma
samples (with a low, a normal and a high hormone concentration
respectively) in each assay; the assay is considered for rejection if by inter-
assay variation the concentration of hormone measured is outside the
range of the mean hormone concentration ± 2 SD of that plasma, as
determined in the previous experiments. The discrepancies between
laboratories one to another may seriously hinder comparison of the results
obtained in different laboratories. Inter-laboratory variation can be
evaluated by the measurement of hormone contents in the same plasma by
different laboratories. Such a program has been initiated in the
Netherlands by the national working group on binding analysis (LWBA).
The results so obtained for the TSH, T4 and T 3 assays however are beyond
the scope of this study.

Last but not least, the most important factor in the validation of a
radioimmunoassay is clinical validation of the assay. That is, the results of
the hormone determinations must make sense. The hormone level must
rise and fall after the addition of known stimulants and suppressants; low
levels must be found in samples from patients known to be deficient in the
 16
hormone, and high levels in those with excessive secretion. The clinical
validation of the assays to be described can be judged by the obtained
results as presented in chapters III and IV.

For the sake of clarity I have adopted the nomenclature for tests of
thyroid hormones in serum as recommended by a committee of the
American Thyroid Association (Solomon, Benotti and DeGroot 1972;
1976). I hope that this nomenclature may be uniformly adopted by all
Dutch thyroidologists and clinical chemists. The nomenclature of peptide
hormones is according to the recommendations of the IUPAC-IUB
Commission on Biochemical Nomenclature (1975). Furthermore, despite
the pamphlet "De blinde mol" (Lindeboom 1971) I used the units of mol
and liter for expression of the measured hormone concentrations where
possible.

References
Berson S A, Yalow RS. Radioimmunoassay. In: Berson S A, ed. Methods in investigative and
diagnostic endocrinology. Volume 2a, Berson S A, Yalow R S, eds. Peptide hormones.
Amsterdam-London: North-Holland Publishing Company 1973: 84-120.
Dijkbaer R. An international recommendation for quantities and units in clinical chemistry.
Ann Int Med 1968; 69: 621-3.
Ekins RP. Basic principles and theory of radioimmunoassay and saturation analysis. Brit
Med Bull 1974; 30:3-11.
Ekins R P. Basic concepts in quality control. In: Radioimmunoassay and related procedures
in medicine 1977, volume II. Vienna: International Atomic Energy Agency, 1978: 6-20.
International Union of Pure and Applied Chemistry — International Union of Biochemistry
Commission on Biochemical Nomenclature. The nomenclature of peptide hormones.
Biochim Biophys Acta 1975; 404: 152-5.
JeffcoateS L. The use of quality control within a laboratory. In: Radioimmunoassay and
related procedures in medicine 1977, volume II. Vienna: International Atomic Energy
Agency, 1978: 57-79.
Lindeboom G A. De blinde mol. Kritische beschouwingen over het door klinisch-chemici
aanbevolen eenhedenstelsel vanuit de kliniek der inwendige geneeskunde. De Erven
F. Bohn N.V.: Haarlem, 1971.
Solomon D H, Benotti J, DeGroot L J. A nomenclature for tests of thyroid hormones in
serum: report of a committee of the American Thyroid Association. J Clin Endocrinol
Metab 1972; 34: 884-90.
The Committee on Nomenclature of the American Thyroid Association. Revised
nomenclature for tests of thyroid hormones in serum. J Clin Endocrinol Metab 1976; 42:
595-8.
Yalow RS, Berson S A. Introduction and general considerations. In: Odell WD, Daugha-
dayWH, eds. Principles of competitive protein-binding assays. Philadelphia and
Toronto, J.B. Lippincott Company, 1971: 1-24.
 17
II.2 Thyrotropin (TSH) ••}
i

The radioimmunoassay of thyrotropin in human blood samples that we '.

have applied, is essentially the method described by Odell and co-workers . *f
(Odell et al 1965). Materials of human origin (human TSH for radioiodina- , |
tion, anti-human TSH serum, purified human pituitary TSH standard) are |
required for maximum assay sensitivity, since the structure of TSH is species- f
specific. TSH antisera react to some extent with other glycoproteins such as , |
follitropin (FSH) and lutropin (LH), since these hormons share a simflar alpha -
subunit. Absorption of TSH antisera with a source of alpha subunits, e.g.
[ choriogonadotropin (CG) decreases crossreactivity to FSH and LH.

"• 2.1. Ingredients.

i a. Buffer-solution. Phosphate buffered saline (PBS) (0.01 M phosphate
\ buffer pH = 7.6, containing 0.15 M NaCl) was used as buffer solution. ;
{ Merthiolate 10"4 M and bovine serum albumin 0.25% (BSA) was added j
to the PBS unless stated otherwise.
b. TSHStandard. As standard served the Medical Research Council's hu-
man pituitary thyrotropin 68/38, which was kindly provided by Dr. D.
Bangham (National Institute for Biological Standards and Control, Lon-
don). It was dissolved and further diluted in PBS, and stored at -20°C.
The approximate estimate of potency by bioassay of this international
reference standard is 3 U/mg (Bangham and Cotes 1971). The loss of
immunopotency during storage at -20°C is low, and has recently been
estimated to be about 0.56% per year (Cotes et al 1978).
c. TSH-Antiserum. Rabbit antiserum to human TSH, preabsorbed with
human choriogonadotropin (hCG), was purchased from Calbiochem,
San Diego, California (lot no. 389523). The lyophilized antiserum was
dissolved in PBS and stored at -20°C in a dilution 1=1000.
d. TSH-Tracer. Human lyophilized TSH was purchased from Calbiochem
(lot no. 389506). lit was solubilized and diluted in PBS (without BSA),
and stored at -20°C in ampoules containing 2 jug TSH in 10 jt/1. Radio- '
labeled thyrotropin was prepared by the chloramine-T method (Green- ;
wood and Hunter 1963). In this procedure phosphate buffer is em- |
ployed to control the pH of the iodination reaction, and the volumes \
are kept low in order to achieve a high percentage incorporation of \ \
12S
I into protein, the yield being strongly dependent on the concen- ,.> ;
tration of the protein. Chloramine-T is used to oxidize the Na 1 2 S I ; J ;
the oxidization is terminated by addition of sodium metabisulphate. ":l -
Carrier protein is sometimes added in larger volumes to stop the iodi- ?|
nation of the peptide hormone and to minimize iodination damage '! 1
(Hunter 1974). } j
In practice our procedure is as follows: 10 /A 0.5 M PBS (pH = 7.6; no i
 18

60 60 70 23 24 25
FRACTION NO. FRACTION NO.

Figure 11.2.
Elution pattern of TSH iodination reaction mixture on Sephadex G-75 fine (column
size 16x1 cm; flow rate 4 drops/minute; fraction volume 0.45 ml). Radioactivity in each
fraction is expressed as counts per minute (c.p.m.).

Figure II.3.
TSH-tracer test of fractions 16-25 (see figure H.2.). Radioactivity of each fraction is
presented in the lower curve, and immunoreactivity in the upper curve (10.000 c.p.m./
50 Ml of each fraction is incubated with 750 MI PBS and 50 y\ TSH-antiserum FD 1=
16.000 for 22 hours at room temperature; separation of the bound hormone fraction by
the DASP technique).

BSA or merthiolate is added to buffer solutions used for iodination),

30 #g chloramine-T in 10 jul 0.5 M PBS, 10 jul 0.5 M PBS and 0.5 mCi
Na * 2 S I in a volume of about 5 #1 (sodium iodide ( 1 2 5 I) for protein
iodination IMS.30, specific activity > 14 mCi/jug, in NaOH solution,
pH 8-11, 100 mCi/ml, purchased from The Radiochemical Centre,
Amersham) are very rapidly added to an ampoule containing 2 ng TSH
in 10 jul PBS; the reaction mixture is swirled continuously. Twenty sec-
onds later 60 jug Na 2 S 2 O s in 20 jul 0.5 M PBS is added, followed by
200 jul 0.2% BSA in 0.5 M PBS fourty seconds after initiation of the
iodination reaction. The reaction mixture is chromatographed on a
Sephadex G-75 fine column (size 16x1 cm), which has been previously
equilibrated with 1 ml 2% BSA in PBS to minimize adsorption of the
12S
I-labeled protein. As elution buffer 0.01 M PBS (pH=7.6) is used,
and fractions of 0.45 ml are collected at a flow rate of 4 drops/minute.
A typical elution pattern is presented in figure II.2. The second peak of
radioactivity (fractions 35AT) is not precipitable with 12.5% trichloro-
acetic acid, and these fractions do not bind specifically to the antibody.
 19
In the first peak radioactivity is precipitated by 12.5% trichloroacetic
acid, and the fractions bind specifically to anti-hTSH antiserum. These
fractions thus contain the radiolabeled thyrotropin; the second peak of
radioactivity constitutes that portion of 1 2 S I not incorporated into
protein. The percentage incorporation of the label into the protein, as
judged from the surfaces of the two peaks, is 30% in this experiment,
and varied from 30 to 57% in four iodinations, with a mean of 43%.
The percent tracer specifically bound to antiserum in the fractions
16-25 of the iodination procedure illustrated in figure II.2, is depicted
in figure II .3. Maximal binding is achieved in fractions at the trailing
edge of the TSH peak, and this is a consistent finding in all our iodina-
tion experiments. We selected the fraction with greatest binding for use
in our assay, and never employed the peak or earlier fractions. The
tracer, diluted in PBS to 10.000 c.pjn./50 fd, was stored at -20°C. The
shelf life appeared to be about 3 weeks. At this time, the second best
fraction obtained at the original gel filtration, was rechromatographed
under the same conditions. The repurified tracer could be conveniently
used for another 3 weeks. Thus, in our hands it sufficed to produce
radiolabeled thyrotropin only once in six weeks.
The specific activity of the tracer, as tested in self-displacement experi- '
ments was on average 130 fiCi/ng, varying from 99 to 161 in six experi-
ments.

2.2. Assay procedure. .

a. Selection of "milieu" in standard curve. To ensure that the milieu in
the tubes containing the standards is as similar to that of the test sam-
ples, it is necessary to add hormone-free serum to the standard tubes.
Thyrotropin-free serum can be obtained from hypopituitary patients, -
from thyrotoxic patients or from subjects pretreated with pharmacolog- I
ical doses of T 3 (T3-suppression sera). :
Alternatively, one can "strip" the hormone from serum, or employ
blood from species whose hormones do not cross-react in the assay
(Golstein and Vanhaelst 1973; Jacobs and Lawton 1974). Sluiter and
co-workers (1972) added 5% bovine y-globulin in buffer in the standard
tubes, which minimized non-specific protein-protein interactions. We '
could confirm then* findings, in that standard curves made in 5% BS7G
equalled the curves in T3-suppression sera, while employing 2% BS7G
or 20% BSA all resulted in a higher percentage of binding. However, we
preferred to use heterologous serum in our homologous test system. Pig ;
i serum could be obtained free of charge from the local slaughter-house. 1
I Figure II.4 demonstrates identical standard curves employing either Jj
I 400 pi T 3 -suppression serum or 400 jul pig serum. The advantage of this '}•
I procedure is its l o w cost. The disadvantage is that w e encountered some j;
i-f
 20
KB
J5

%B

6 8 10
AlU TSH/lube reciprocal final dilution antiserum

Figure 11.4,
TSH-standard curve, made in human T3-suppression serum (•) or pig serum (x). Reac-
tion conditions: SO jul standard, 400 M! serum, 50 nl tracer, 50 fil AS (FD 1-16.000) and
buffer up to 800 Ml are incubated for 4 days at 4°C; separation by DASP.

Figure 11.5.
TSH-antiserum dilution curve (incubation of antiserum and 50 jul TSH* in buffer for 4
days at 4°C; separation by DASP).

differences from time to time with serum from different pigs. There-
fore each new batch of pig serum has to be checked carefully. Finally,
non-specific binding of tracer varies from one plasma sample to an-
other, and therefore one may occasionally find a percentage bound
greater than the "initial binding", i.e. the percentage of tracer bound to
the antibody in the first zero-standard point of the standard curve.
b. Selection of tracer concentration. When the reaction mixtures were
incubated for 22 hours at room temperature, standard curves employ-
ing SO jul TSH* (10.000 c.p.m.) showed higher initial binding and a
steeper slope as compared with curves using 25 or 100 JJI TSH*. HOW
ever, nearly identical standard curves were obtained with 25, SO or
100 ptl TSH* when the incubation was performed for 4 days at 4°C.
Since we decided to incubate for 4 days at 4°C (see below), any
one of these tracer volumes could be used. For reasons of pipetting
convenience, counting time efficiency and tracer economy the volume
of SO JLCI TSH* was chosen. It is calculated from the self-displacement
experiments that SO jul radiolabeled thyrotropin contained on average
0.16 MU TSH.
c. Selection of antiserum dilution. Tracer binding to the antiserum at
various dilutions is depicted in figure II .5. For economical reasons
binding of the tracer with undiluted antiserum was not tested. Three
 21

7 .i
Si
—incubation 24hours, room tetnp
4days .

6 8 10
JUU TSH/lube

FjgareJT.6.
TSH-standard cuives under different incubation conditions. Reaction mixture: 50 »xl
tracer, SO pi AS (FD 1=16.000), 400 jul pig serum, SO p\ standard, buffer up to 800 Ml;
separation by DASP.

different dilutions of antiserum were tested, i.e. final dilutions (FD)

of 1=8000, 1=12000 and 1=16000. Although the initial binding was
highest in FD 1=8000 and lowest in FD 1=16000, the slope of the
standard curves employing a FD 1=16000 was the steepest, and conse-
quently sensitivity was greatest at this final dilution. This was true un-
der the two incubation conditions tested, i.e. 4 days at 4°C, or 22 hours
at room temperature. Sensitivity decreased at final dilutions of anti-
serum 1=20000 and greater.
d. Selection of incubation conditions. We compared a long incubation (4
days at 4°C) with a short incubation (22 hours at room temperature).
Sensitivity was consistently greater under the longer incubation con-
ditions (figure II.6), and this was true for the three different concen-
trations of both tracer and antiserum tested. Therefore 4 days at 4°C
was chosen. Preincubation in the absence of tracer (which in some
radioimmunoassays is known to increase sensitivity) during one or more
days did not increase sensitivity substantially.
e. Separation of bound and free hormone. The double-antibody solid-
phase method was chosen as separation technique. Immunological
precipitation of bound fraction is achieved in this method by sheep
anti-rabbit 7-globulin antibodies coupled to cellulose (anti-rabbit
DASP®, Organon, Holland). Tween 20 (0.15% v/v) was added to reduce
aggregation of micro-particles and non-specific adsorption of tracer
(Ratcliffe 1974).
f. Assay procedure. The assay procedure as finally determined, starts with
t- 22
pipetting consecutively the following reagents in 4 ml polystyrene
tubes:
x Ml PBS up to a final volume of 800 JKI
10-50 Ml standard (hTSH, MRC 68/38) in standard curve tubes only (0.5-10 MU
TSH per tube).
400 MI unknown plasma sample, or pig serum in standard curve tubes
i
50 Ml tracer, containing ± 10.000 cp.m. ~ 0.16 iiV TSH.
5c 50 Ml antiserum 1=1000(rabbit anti-hTSH,Calbiochem), final dilution 1=16000
?••:
After mixing, the tubes are incubated at 4°C for 4 days. Then, to each
test tube 1 ml of DASP® suspension (containing one 5 ml vial DASP,
Tween 20 0.15% v/v and PBS up to 50 ml) is added followed by rotat-
ing incubation at 4°C for 24 hours. The antibody-bound hormone frac-
tion is collected by centrifugation, the supernatant being sucked off,
and the radioactivity in the pellet is counted after a single wash with
physiological saline in a gamma-scintillation camera.
All samples are also incubated in the absence of antiserum (control
tubes or "blanks") in order to correct for non-specific binding of the
tracer. Standards are run in triplicate, unknown plasma samples in
duplicate.

2.3.Results.
a. Sensitivity and precision.
The precision of the standard curve was determined by the assay of
each standard point in ten-fold in one experiment. The results are given
in table U.I and figure II.7. Sensitivity is 0.10 uU TSH/tube, or 0.25
%B
25

0 12 4 6 8 10
UU TSH/luta

Figure 11.7.
Sensitivity and precision of the TSH-standard curve (the mean ± 2.SD of the standard
points, each assayed in tenfold, is depicted).
 23
Table ILL
Precision of the TSH standard curve (each point assayed in tenfold).
standard point percentage bound coefficient of variation
0*U TSH/tube) mean SD (^°-x 100%) ft
f
0 23.84 0.39 1.64
O.S 20.16 0.34 1.69
1 17.35 0.39 2.25
2 13.32 0.39 2.93
4 9.98 0.31 3.11
6 8.01 0.27 3.37
8 6.75 0.34 5.04
10 6.16 0.20 3.24

mU/1 plasma. The detection limit of the assay therefore is rounded off
at 0.3 mU/1 plasma. There is no complete discrimination between the
last two points of the standard curve. Plasma samples with a TSH con-
centration of > 20 mU/1 (i.e. > 8 juU/tube) must consequently been
diluted and assayed again. The precision of measuring TSH concentra-
tion in unknown plasma samples is presented in table II .2. Intra-assay
variation is lowest at hormone levels between 2 and 8 mU/1, i.e. meas-
urement seems to be most precise at hormone levels of 0.8 to 3.2 juU/
tube,
b. Specificity.
Non-specific adsorption in the assay system (binding of tracer in the
absence of antibody) was low, and seldom exceeded 3%. Similarity be-
tween standard curves and dilution curves of plasma samples could be

Table 11.2.
Intra-assay variation of unknown plasma samples in the TSH radioimmunoassay (each
sample assayed in tenfold).
unknown plasma TSH mU/1 coefficient of variation
mean SD (^-x 100%)

A 1.8 0.11 6.11

B 2.0 0.08 4.00 ..,'•3

C 2.9 0.11 3.79

D 3.0 0.18 6.00
E 8.0 0.36 4.50
F 10.5 1.21 11.52
G 21.8 3.02 13.85
 24
%B jui unknown ptawna %6 «l unknown plasma
SO tOO 200 50 100
30 30

4 6 8 2 4
juU TSH/lube uU TSH/lube

Figure II.8.
Paiallelism between standaid cuive and dilution cuive of unknown plasma sample in the
TSH ladioinununoassay.

Figure II. 9.
Parallelism between standaid cuive and dilutions of unknown plasma samples (taken be-
fore and after TRH stimulation in a patient) in the TSH radioimmunoassay.

0.S 8 10 20 40 80100 hFSH.hLH ng/tube

40 100 200 400 1000 2000 4000

%B
30

24

18

12

hTSH

0.5 4 6 8 10 20 100 hTSH jiU/tUbe

Figure n.10.
:\ Cross-reactivity of hCG (Pregnyl®, (Organon, Holland), hLH and hFSH (butt. CPDS/2,
obtained by courtesy of Dr, Lequin and Dr. Hennen) in the TSH radioimmunoassay
(50% displacement of the TSH tracer is effectuated by 0.66 ngTSH/tube (2 (iV), or by
5ngFSH/tube).
 25
Table 11.3.
Expected and found concentrations of TSH (mU/1) in recovery experiments with three
different plasma samples.
added TSH (mU/1)
0 1 2 4 6 8

plasma K: expected 1.4 2.4 4.4 6.4 8.4

found 0.4 1.3 2.55 4.3 6.15 10.1
% recovery 93 106 98 96 120
plasma L: expected 2.05 3.05 5.05 7.05 9.05
found 1.05 2.05 3.4 4.8 6.9 9.9
% recovery 100 111 95 98 109
plasma M: expected 3.55 4.55 6.55 8.55 10.55
found 2.55 3.45 4.1 7.0 10.0 9.0
% recovery 97 90 107 117 85

demonstrated (see figures II.8 and II.9), i.e. there was no obvious
deviation from parallelism between these lines. Hormonal cross-reactiv-
to ity was tested for the related human glycoproteins choriogonadotropin,
i follitropin and lutropin (figure 11.10). The preparation of follitropin
used in this experiment reacts to a considerable extent in the TSH
assay, and in the high concentration range of FSH parallelism is ob-
served with the TSH standard curve. Plasma TSH concentration was
measured in 10 postmenopausal and in 10 premenopausal women;
there was no difference between the two groups (1.0 ±1.0 vs. 1.0 ±
0.5 mU/1). The recovery of exogenously added TSH to plasma samples
with a known TSH concentration is given in table II.3. The mean
percentage recovery in these fifteen experiments is 101.5%.
c. Quality control.
The coefficient of inter-assay variation is about 10% (see table II .4).

Table 114.
Inter-assay variation of plasma pools in the TSH radioimmunoassay (n - number of assays).
plasma pool TSH mU/1 coefficient of variation
mean SD (SJ2 x 100%)

P 18 1.6 0.17 10.43

Q 14 1.9 0.16 8.42
R 19 2.3 0.25 10.87
S 16 7.4 0.85 11.49
T 6 27.0 2.97 11.00
 26 - t
2.4. Discussion. ^
••iz
The reaction conditions chosen in our TSH radioimmunoassay were die- H
tated by the goal of reaching maximal sensitivy. The slightly better sensitivity . }i|
achieved byPekaryetal. 1975(0.2mU/lvs.025mU/linourassay)seemsmainly [ « fe«
due t o the relatively high affinity constant of their hTSH antibody, which per- ' P?
mits a final dilution of the antiserum of 1=1.000.000 in their assay. Preparation I K
and selection of optimally radiolabeled TSH is of critical importance for the I ; £='•.
sensitivity and validation of the TSH assay. Although it has been described j ••-' |;
that human 12 s I-labeled TSH prepared by lactoperoxidase shows less damage b
on storage, an increased shelf-life, and improved binding at different anti- r
serum dilutions as compared to chloramine-T labeling (Burrin 1976), all ;•
authors reporting high sensitivity in the TSH assay use chloramine-T labeling; fc
we have used this method exclusively. Thyrotropin tracers of high specific , \.
activity are less immunoreactive and consequently decrease sensitivity as ;.
opposed to tracers of low specific activity (Erhardt et al 1973; Moser and j |
Hollingsworth 1975). Tracers of low specific activity however decrease count- i
ing efficiency. Therefore as a compromise iodination conditions were chosen ; |
resulting in radiolabeled TSH of intermediate specific activity (specific I
activities are in our assay ± 1 3 0 ^Ci/jug, and 220 -123 - 43 in the experiments
of Erhardt et al). The shelf-life of our tracer is in the same order of magni-
tude as those reported by others (Hershman and Pittman 1971: 2 months if
repeatedly purified by gel filtration; Erhardt et al 1973: 3 weeks without
purification; Burrin 1976: 3 weeks). We find maximal binding to the anti-
body in tracer fractions from the trailing edge of the TSH* peak. This is in
agreement with Gordin and Saarinen (1972) who report that selection of a
specific fraction after gel filtration of the iodination mixture can be critical
for the sensitivity and validation of the TSH assay. Thus, parallelism between
the standard curve and a dilution curve of a serum sample could be demon-
strated using fractions from the trailing edge of the TSH* peak; however
parallelism disappeared when the peak fraction was used (Jacobs and Lawton
1974). This phenomenon may be explained by the presence of damaged
components of the labeled peptide hormone, which due to their frequent
binding to proteins, particular albumin and a-globulins, elute earlier than the
purified labeled hormone (Berson and Yalow 1973).
Optimal incubation conditions have been investigated in detail by Erhardt
et al (1973). Equilibrium was not reached in his experiments at any of the
temperatures under test (4 - 12 - 20 - 28 - 32 - 37°C), even not after 140
hours incubation. Initial binding was highest at 20°C incubation, and reaction
rate at this temperature was twice as great as at 4°C. Non specific binding
was 2.5%, being constant for all temperatures tested. In contrast, maximal
binding was the same at each temperature ( 4 , 2 0 and 37°C) and in all groups
equilibrium was reached after 4 days in the experiments of Gordon and
Table 115
Main features of TSH radioimmunoassays, as reported in literature.

REFERENCE TRACER ANTISERUM STANDARD INCUBATION CONDITIONS SENSITIVITY NORMAL VALUES mU/l
spec. act. pg added F.D. pro separation
MCi/jug in assay incubation incubation BandF mU/l n mean ± SD range

Hersman 1971 1=175.000 Int. Human 5 days DA 1.25 173 3.9 ± 2.0
TSH Standard 5"C
Patel 1971 150-400 25 1=300.000 MRC A 24 hours 4 days DA 0.4 69 1.0 <0.4-3.1
4°C 4°C
Hall 1971 200-300 100 1=600.000 MRCA 48 hours 5 days DA 0.5 29 1.6 ± 0.8 <0.5-4.2
4°C 4°C
Sluiter 1972 ± 150 1=4.000 MRC 68/38 48 hours 5 days DASP 0.5 37 2.0 ±1.53
4°C 4°C
Gordin 1972 ± 100 1=300.000 MRCA DA 1.0 71 3.6 ± 1.4 1.6-8.8
Erhardt 1973 36 1250 1=180.000 MRC 68/38 44 hours 2.5 days DA 0.7 0-3.8 4
18-25°C 18-25°C
140 hours 2.5 days DA 0.4
Moser 1975 35 MRC 68/38 72 hours 4 days DA 29 5.7 2.8-11
Wood 1975 48 hours 5 days DA 0.4 80 1.9 <0.5-5.3
140-160 30 1=40.000 MRC 68/38
4°C 4°C
I
i
6 hours 1 day DA 0.4
'•
37°C 37°C
Pekary 1975 41-49 1=1.000.000 MRC 68/38 36 hours l.Sday DA 0.2 54 1.5 ± 1.0
4°C 4°C
Nisula 1978 150-250 1=60.000 MRC 68/38 5 days DA 0.56 9 <0.56-4.0
4°C
Wiersinga 1979 ±130 ±53 1=16.000 MRC 68/38 4 days
4°C
DASP 0.25 63 1.2 ±1.1 <0.3-4.5
j
 "3—

28
Saarinen (1972). In our hands, incubating at 4°C for 4 days resulted in
higher initial binding and greater sensitivity as compared to incubation at
20°C for 22 hours, and was therefore preferred. Preincubation by adding
tracer hormone at a later time may increase sensitivity, and this procedure
can be of value for the TSH assay as reported by Erhardt et al (1973): sen-
sitivity at preincubation times of 0, 44 and 140 hours was respectively 2.0,
0.7 and 0.4 juU/ml. Most authors claiming great sensitivity of their TSH
assay, employ preincubation without tracer (see table II.5). Preincubation
in our system however did not increase sensitivity consistently to an extent
which warranted routine use of this time consuming procedure, and was
therefore discarded. Separation of bound and free hormone was performed
by the double-antibody solid-phase technique. We found this method as
reliable and easy to handle as Sluiter et al (1972). The time required for pre-
cipitation of the bound hormone fraction by the second antibody may be
much shorter than 24 hours (Erhardt et al 1973), but this point was not
further investigated.
Thyrotropin, follitropin, lutropin and choriogonadotropin are structurally
related glycoproteins, each composed of an a- and 0-subunit. The chemical
structure of the a-units are very similar in these four peptides, the /3-units
giving the molecule its specific biological properties. Consequently, hCG,
hLH and hFSH may cross-react in the hTSH-RIA to a considerable extent.
Cross-reaction to these peptides may be prevented by adding hCG to the test
tubes or preadsorption of the antiserum with hCG (Odell et al. 1965). Never-
theless, we observed strong interference of FSH in the TSH assay, apparently
due to cross-reactivity. The parallel path of the FSH curve and the TSH
standard curve, however, indicates contamination of the FSH preparation
with TSH rather than real cross-reaction to FSH. This possibility is even more
likely in the light of reports about the hTSH contamination of "immuno-
chemical grade" hFSH preparations, which in one case was 22% by weight
(Parlow 1975). The most convincing evidence that FSH does not cross-react
in our TSH assay to any extent of clinical importance, is the absence of
differences in TSH levels between postmenopausal women (with high FSH
plasma concentrations) and premenopausal women.
The described TSH assay is peculiar in its use of a relatively high concen-
tration of plasma (400 jil in a final volume of 800//!), by which non-specific
effects may arise more frequently. In practice this has not been encountered.
The sensitivity of the assay decreases to 0.5 mU/1 plasma if 200 jul plasma is
used.

The main features of some TSH radioimmunoassays described in the

literature are presented in table II.5. We may conclude that our TSH assay is
very sensitive, precise and meeting the requirements for qualitative and
quantitative specificity and good quality. Although most euthyroid individ-
 29
uals have detectable TSH plasma levels and most hyperthyroid patients have
not, there is some overlap in the radioimmunoassay of TSH between the
euthyroid and hyperthyroid range for plasma TSH levels (Hall 1972). Since
sensitivity seems to be near its maximum in the reported radioimmunoassays
of TSH, other techniques in which sensitivity can be dramatically increased
are needed for complete separation of euthyroid and hyperhyroid subjects in
the TSH assay, A newly designed cytochemical bioassay for TSH based on
quantitative microhistochemistry (measuring the TSH-induced increase in
lysosomal membrane permeability in thyroid follicle epithelial cells) is
promising in this respect, since this assay is 104 times more sensitive than
current radioimmunoassays (Bitensky et al (1973). Nevertheless, with use
of this assay the overlap in TSH values of euthyroid and hyperthyroid
individuals is still present (Petersen et al 1975).

2.5. References.
Bangham D R, Cotes P M. Reference standards for radioimmunoassays. In: Kirkham K
E, Hunter W M, eds. Radioimmunoassay methods. Edinburgh and London: Churchill
Livingstone, 1971: 345-68.
Berson S A, Yalow R S. Radioimmunoassay. In: Berson S A, ed. Methods in investigative
and diagnostic endocrinology. Volume 2a, Berson S A, Yalow R S, eds. Peptide
hormones. Amsterdam - London: North-Holland Publishing Company 1973: 84-
120.
Bitensky L, Alaghband-Zadek J, Chayen J. Studies on thyroid-stimulating hormone and
the long-acting thyroid stimulating hormone. Clin Endocrinol 1973; 3: 363-74.
Burrin J M. Comparison between human' * s I-labelled TSH labelled with lactoperoxidase
or chloramine-T; advantages in the use of enzymically labelled antigen in a radioim-
munoassay for human TSH. Clin Chim Acta 1976; 70:153-9.
Cotes P M, Gaines Das R E, Kirkwood T B L, Bennie J G, Hunter W M. The stability of
standards for radioimmunoassay of human TSH: research standard A and the inter-
national reference preparation initially 68/38. Acta Endocrinol 1978; 88: 291-7.
Ergardt F, Marschner I, Pichardt R C, Scriba P C. Improvement and quality control
of the radioimmunological TSH determination. J ClinChem Clin Biochem 1973; 11:
381-7.
Golstein J, Vanhaelst Z. Influence of thyrotnrain-free serum on the radioimmunoassay
of human thyrotropin. Qin Chim Acta 1973;49:141-6.
Gordin A, Saarinen P. Methodological study of the radioimmunoassay of human thyro-
tropin. Acta Endocrinol 1972; 71: 24-36.
Greenwood F C, Hunter W M, Glover J S. The preparation of 1 3 1 I-labelled human
growth hormone of high specific radioactivity. Biochem J 1963; 89:114-23.
Hall R, Amos J, Ormston B J. Radioimmunoassay of human serum thyrotropin. Brit
MedJ 1971; 1:582-5.
Hall R. The immunoassay of thyroid-stimulating hormone and its clinical applications.
Clin Endocrinol 1972; 1:115-25.
Hershman J M, Pittman Jr J A. Utility of the radioimmunoassay of serum thryrotrophin
hi man. Ann Int Med 1971; 74:481-90.
Hunter W M. Preparation and assessment of radioactive tracers. Brit Med Bull 1974; 30:
18-23.
 •~n—

- - 3 0 •"-,._:-.:,...=
Jacobs H S, Lawton N F. Pituitary and placental glycopeptide hormones. Brit Med Bull
1974; 30: 55-61.
Moset R J, Hollingsworth D R. Examination of some factors affecting sensitivity and
reproducibility in radioimmunoassay of thyrotropin. Clin Chem 1975; 21:347-52.
Nisula B C, Louvet J P. Radioimmunoassay of thyrotropin concentrated from serum.
I Clin Endocrinol Metab 1978; 46: 729-33.
Odell W D, Wilber J F, Paul W E . Radioimmunoassay of thyrotropin in human serum.
J Clin Endocrinol Metab 1965;25: 1179-88.
Parlow A F. The immunoreactive hTSH contamination of "immunochemical grade"
, hFSH preparation. J Clin Endocrinoi Metab 1975; 41: 189-90. -
Patel Y C, Burger H G, Hudson B. Radioimmunoassay of thyrotropin: sensitivity and
specificity. J Clin Endocrinol Metab 1971; 33: 768-74.
Pekary A E, Hershman J M, Parlow A F. A sensitive and precise radioimmunoassay
for human thyroid-stimulating hormone. J Clin Endocrinol Metab 1975; 41: 676-84.
Petersen V, Smith B R, Hall R. A study of thyroid stimulating activity in human serum
with the highly sensitive cytochemical bioassay. J Clin Endocrinol Metab 1975; 41:
199-202.
Ratcliffe J G. Separation techniques in saturation analysis. Brit Med Bull 1974; 30:
32-7.
Sluiter W J, Kersen F van, Zanten A K van, Beekhuis H, Doorenbos H. A radioimmuno-
assay of human TSH, employing a solid phase second antibody, and a purified
globulin preparation for standardization of nonspecific protein interactions. Clin
Chim Acta 1972; 42: 255-62.
Wood P J, Smith J, Marks V. Notes on the radioimmunoassay of human thyrotropin.
Ann clin Biochem 1975; 12:196-9.
 31
v. i
£.? //.J. Iodothywnines. ?
fl |
§" The chemical structure of the various iodothyronines differs only in the f|
I number and/or position of the iodine atoms. Due to the similarity in chemical |
'•• structure it is not surprising that so-called "specific" antisera raised against a • .. ' |
f particular iodothyronine may cross-react considerably with other iodothyro- • I;
I nines. With the techniques cunently available an absolute specific antiserum £
directed against just one iodothyronine cannot be obtained; only the condi- ; [-'.
tions chosen for the performance of a radioimmunoassay make the assay -j p
rather specific for the iodothyronine under test. This is illustrated by the |
following set of experiments. !
; Radiolabeled iodothyronines, either commercially bought ( 12S I-T 4 and jt
12 S
I-T 3 , Abbott Laboratories) or self prepared ( 1 2 s I-rT 3 by iodination of f.
3,3'-T2; for details see below), were purified by Sephadex G-25 fine chroma-
i. tography (column size 40 x 2.5 cm, volume 135 ml; elution buffer 0.01 M
i NaOH in distilled water pH=12; fractions of 2.85 ml each were collected at a ;
flow rate of 4 drops/min). The radioactive elution patterns are depicted in
figure n.l 1. Two comments may be made. Firstly, pure tracers of T 4 , and T 3
and rT 3 are easily obtained by chromatography of the iodination mixture.
Secondly, iodothyronines with a higher molecular weight have a larger elution
volume than those with a lower molecular weight, and different iodothy-
ronines with the same molecular weight (T 3 and rT 3 ) elute at different
volumes. Therefore Sephadex chromatography separates iodothyronines not
by gel filtration (a process in which larger molecules elute before the smaller
ones) but by differential adsorption of iodothyronines onto the gel. From
these and other experiments using smaller columns, we calculated the relative
retardation of iodothyronines by Sephadex. Mean ratios V x /V t (V x = elution
volume of substance x; V t = total volume of the column) are: * 25I-albumin
0.39, 1 2 S I 0.66, 1 2 s I-3^'-T 2 1.33, 1 2 S I-T 3 1.70, 1 2 S I-rT 3 2.97, I 2 S I-T 4
3.88. The adsorption of iodothyronines onto the Sephadex gel is illustrated
by the fact that the ratio V x /V t is > 1. It has been reported that column
adsorption may be influenced by the phenolic hydroxyl group and by the
number and location of iodine atoms in the iodothyronine molecule: in gen-
eral more retardation by the gel is observed at a greater number of iodine
atoms, and less iodine in the outer ring is associated with a smaller elution
volume (Lissitzky et al 1962; Mougey and Mason 1963; Blasi and De Masi ;
1967). •
The purified tracers were incubated separately with various dilutions of
the best (i.e. most specific) antisera against T 4 , T 3 and rT 3 . Tracer hormones I
were added at volumes of 50 jil (10.000 c.pjn.), containing either 32 pg T 4 , I
85 pg T 3 or 4 pg rT 3 . Antisera were raised in rabbits against bovine thyro- %
:
globulin (T 4 -antiserum 3045), against a conjugate of T 3 -bovine thyroglobulin :
(T3-antiserum 137) and against a conjugate of rT 3 -BSA (rT 3 -antiserum 3686) I
 32
1.0

- V,
If 0.5
ri.
d

. .-7

I- T2*
2.0
o
X

15

1.0 •

IJ
0.5

n
-

0 20
J
40 60 80 100 120 140 160 180 200
FRACTION NO
Figure n.lL
Purification of ll5 Mabeled T4 (top), T s (middle) and rT, (bottom) by Sephadex
chromatography (for details see text).

•rrry
 33

T4- antiserum 3045

f.r

101 103 TO"

T3-antiserum 137

104

rT 3 -antiserum 3686
100 r

90

80

70

60

50

40

30

20

10

10Z 103
reciprocal final dilution antiserum
Figure 11.12.
Binding of radiolabeled T 4 , T, and rT3 to each of the so-called "specific" antiseia used
in the radioimmunoassay of T 4 , T , and rT3 respectively.
 34
(for further details see below). It is obvious (figure 11.12) that each "specific"
antiserum also binds the other two iodothyronines, albeit at a higher concen-
tration of the antiserum. All three iodothyronines are present in thyro-
globulin, and consequently immunisation by thyroglobulin raises polyclonal
antisera. However, the rT3-antiserum also cross-reacts with T4 and T 3 . There-
fore, these antisera are not really specific, and in principle each of these
three antisera might be used in the assays of both T 4 , T3 and rT 3 , if the
appropriate dilution is chosen. In the assay of plasma iodothyronines this is
not always practical, because the molar concentration of rT3 in human
plasma in 8 times less than of T 3 , and 460 times less than T 4 . Therefore, the
radioimmunoassay of rT3 is the most critical one, and will be discussed in
detail. The same difficulties and procedures apply to the T3 and T4-RIA's,
and these assays will be described only briefly.

11.3a. Reverse triiodothyronine (rT3).

3a. 1. Ingredients.
a. Buffer-solution. The composition of the buffer is: sodium barbital 0.075
M, merthiolate 10"4 M, bovine serum albumin 0.25% in distilled water,
pH=8.6. Sodium barbital is added to block the binding of iodothyronines
to prealbumin and albumin, merthiolate to prevent bacterial growth, bo-
vine serum albumin to prevent adsorption of proteins, and 1 M HC1 as
much as needed for reaching a pH=8.6 since the antigen-antibody reaction
for iodothyronines in general is optimal at an alkaline pH below 9 (Hufner
and Hesch 1973). Alkalinization also inhibits binding of iodothyronines to
plasma proteins. Unless stated otherwise this buffer is used throughout the
assays of iodothyronines.
b. rT3-standard. 3,3',5'-triiodo-L-thyronine (rT 3 , lot no. 545/24B) was ob-
tained by courtesy of Henning Berlin GmbH. Contamination of this agent
with T3 and T4 is < 0.1%. Standard preparations were made by dissolving
rT3 in 10 ml 0.01 N NaOH/propylene-glycol (1:2) solution, and further
dilution in buffer. Storage was at -20°C.
c. rT3-antiserum. Six white rabbits ("Vlaamse reuzen") were injected intra-
muscularly at four sites on the thighs and the back with 0 J 5 mg rT3 -BSA
conjugate (courtesy of Dr. H. Steinmaus, Henning Berlin GmbH) in a
suspension of physiological saline in complete Freund's adjuvant (l:3,v/v).
A mixture of saline, incomplete and complete Freund's adjuvant
(l:1.5:1.5,v/v) was used at the second and following immunizations,
which took place at three week intervals. Blood was withdrawn two weeks
after the third and fourth immunization. All rabbits developed antibodies
to rT3 of good avidity. However, cross-reaction to.T4 was in no case lower
than 0.17%, which we considered inacceptably high. Absorption of anti-
sera with T 4 decreased crossreactivity to 0.08%, but was associated with
 35
7ViWe//.6.
Tlieorctical maximal specific activity for radioiodothyronines.

number o f ' 2 * 1 atoms/molecule

I 1 2
ft-
3000 6000
3500 7000
3500
4400

considerable loss in avidity and sensitivity. We are most grateful to Dr.

T.J. Visser (Rotterdam) who donated rT3-antiserum with a lower cross-
reactivity to T 4 ; this antiserum (no. 3686) is also raised in rabbits against
the rT3-BSA conjugate of Henning (Visser, Docter and Hennemann 1977).
d. rTS-tracer. Tracers of high specific activity are required for the develop-
ment of sensitive assays for iodothyronines. The theoretical maximal
specific activity for radioiodothyronines (according to Kochupillai and
Yalow 1978) is given in table II.6. Radioiodothyronines can be produced
by exchange of iodine atoms, which results in lower specific activity
tracers, and by addition of iodine atoms, which results in a very high
specific activity (Nakamura, Chopra and Solomon 1977; Kochupillai and
Yalow 1978). Addition of an iodine atom to the outer-ring occurs much
more frequently than exchange labeling of outer-ring iodine atoms. There-
fore, outer-ring radiolabeled iodothyronines of high specific activity are
easily prepared when the substrate for iodination has iodina atom less in
the outer ring than the compound desired (Nakamura, Chopra and Solo-
mon 1977; Kochupillai and Yalow 1978). Thyronines labeled in the inner
ring of the molecule have never been encountered in these procedures.
Consequently, radiolabeled rT 3 is prepared in our laboratory as follows.
Volumes of 20 jul 0.5 M phosphate buffer pH=7.5,20 ;ul 0.5 M phosphate
buffer containing 10 /ig chloramine-T (freshly made) and 10 jul 1 2 S I (1
mCi) are very rapidly added to 0.5 jug 3,3 '-T2 (obtained from Henning Ber-
lin GmbH, dissolved in 0.05 M phosphate buffer pH=7.5); the iodination
reaction is stopped after 30 seconds by addition of 100 jug ex tempore
prepared Na 2 S 2 O s in 20 jtd 0.5 M phosphate buffer and the reaction mix-
ture is applied on a small Sephadex LH-20 column of 1.5 ml. Unreacted
12 s
I is removed by washing the column in succession with 1 ml of 0.05 M
phosphate buffer, 4 ml of distilled water and 1.2 ml of 99.1 (methanol):
 36
(2N NH4OH). The radiolabeled iodothyronines are than eluted with 4 ml
of 99:1 (methanol): (2N NH4OH), and the eluate is dried under a stream
of nitrogen. The residue is redissolved in 1 ml of propylene glycol - 0.01
M NaOK (2:1), and chromatographed on a Sephadex G-25 fine column,
size 15x1 cm. The elution buffer is 0.05 M phosphate buffer pH=7.5, and
the fraction volume 3ml. The elution profile consists of three peaks,
provisionally identified as iodine 3,3'-T2 and rT 3 . The best fractions of the
rT3-peak are pooled, diluted in buffer to 10.000 c.pjn./25 jul, and stored
at -20°C.
In three successive iodinations specific activity of 1 2 S I-rT 3 was found
to be 3070,3024 and 3385 juCi/jug. These values approximate the theoreti-
cal maximum. In our hands the shelf life of the rT 3 is about 10-12 weeks.

310.2. Assay procedure.

a. Selection of milieu. Thyroid hormones are transported in plasma by
;j
* thyroid hormone binding proteins (TBP), and over 99% of thyroid hor-
mones circulating in the human plasma is bound to these proteins. In
determining thyroid hormone content of human plasma samples, the
binding capacity of TBP must be neutralized in order to prevent
competition between TBP and antiserum for thyroid hormones. This can
be done by extraction of the thyroid hormones (by ethanol as applied to
the rT 3 assay by Chopra (1974) and Meinhold (1975), or most elegantly
by extraction of iodothyronines onto small Sephadex columns in an
alkaline medium as described for rT 3 by Faber et al in 1978). If one wants
to avoid the more laborious extraction procedures, it is necessary to block
the binding of iodothyronines to TBP in all tubes. This can be done by
heating (Sterling and Milch 1974), by alkalinization up to a pH > 11.0
(Hiifner and Hesch 1973), or by addition of agents which displace iodo-
thyronines from their binding sites on TBP (so-called blocking agents).
We preferred to avoid extraction procedures, and proceeded as follows.
1. Preparation of iodothyronine-free plasma.
Iodothyronine-free plasma is prepared by repeated washings of a human
plasma pool with the resin AG 1-X2 (Analytical Grade Anion Exchange
Resin AG 1-X 2 ,200400 mesh chloride form, Bio-Rad catalog no.
1401251). The procedure in detail is : 200 gram of AG 1-X2 is washed
consecutively ten times with 600 ml of distilled water, three times with
400 ml 0.225 M sodiumbarbital pH=8.6, and ten times with 0.075 M
sodiumbarbital pH=8.6; 500 ml of pooled human plasma is mixed with
50 gr. AG 1-X2 under rotating conditions for one hour; after centrifu-
• [
I gation the same procedure is applied to the supernatant for another three
\ times. The effectiveness of the procedure is checked by performing ex-
l pertinents in which known amounts of radiolabeled iodothyronines are
 37
%B
60

m
%B
70

Q5 1.0 290 500

jjg M I S / t u b a
fct

Figure 11.13.
rTj -standard curve, made in iodothyronine-free plasma prepared by the resin technique
(•) and in plasma of a severely hypothyroid patient (o). Reaction conditions: 50 jil
z1 standard, 50 it\ ANS (300 ftg), 50 jul plasma, 50 nl antiserum (FD 1=100.000), 25 nl
rTs *, 775 ii\ buffer; incubation for 5 days at 4°C; separation by PEG.

Figure 11.14.
Initial binding of rT, as function of 8-anilino-l-naphthalene sulphonic acid (ANS) con-
centration in the rT,-assay (in the presence of iodothyronine-free plasma).

added to the plasma pool. We found that this procedure removed 99.2% of
the rT3-tracer, 99.5% of the T3-tracer and 99.3% of the T4-tracer, that is
the prepared plasma contains less than 1% of its original iodothyronine
content. In our hands the resin technique was easier to handle and slightly
more effective than stripping the plasma pool with charcoal. Standard
curves made in iodothyronine-free plasma prepared by the resin technique,
or in plasma from a severely hypothyroid patient supposed to have un-
detectable plasma levels of rT3 (figure n.13) were superimposable, and
this is accepted as further proof of the validity of the procedure.
2. Selection ofTBP-binding blocking agent.
Several agents which block the binding of iodothyronines to TBP have
been used in the assays of iodothyronines, especially salicylate (Larsen
1972), merthiolate (Hufner and Hesch 1973) and 8-anilino-l-naphthalene
sulphonic acid (ANS) (Chopra 1972; Mitsuma et al 1972). Salicylate is
mainly used in the radioimmunoassay of T 3 , since very large quantities are
needed for full dissociation of T 4 from its binding to TBP. It is reported
that merthiolate is equally effective (Hufner and Grussendorf 1976) or less
 38

effective (Ratcliffe et al 1976) than ANS in blocking rT3-binding to TBP.

Since we were already accustomed to the use of ANS in the T4-assay, we
therefore decided to use ANS also in the rT3 -assay.
Figure 11.14 depicts initial binding of rT 3 * as a function of ANS concen-
t-
r-f. tration in the incubation mixture; the experiments were performed in the
presence of iodothyronine-free plasma. Clearly, without the addition of
ANS the labeled hormone fraction specifically bound to the antibody is
low, since the tracer is not prevented to bind to the TBP in the iodo-
thyronine-free plasma. At concentrations of 31.2 and 62.5 /ag ANS/tube
the percent hormone bound to antibody is highest, whereas a decrease is
seen with increasing ANS concentrations. Thus, ANS in high concentration
decreases the binding of tracer to antibody in the rT3-RIA. Since in plas-
mas with high TBG levels (e.g. pregnant women, subjects on estrogen
medication) the lower concentrations of ANS did not completely block
the rT3-TBP binding, we routinely used 300 jug ANS/tube. However, at
very high TBP-concentrations no complete blocking of rT3-TBP binding
may occur.
b. Selection of tracer concentration. Due to the high specific activity of the
radioiodinated rT 3 , quantities as low as 0.00312 pmol or 2 pg rT3*/tube
can be used in a volume of 25 jul (10.000 cpm.) The amount of tracer
hormone added is two times less than the hormone concentration of the
first standard point (0.0062S pmol or 4.06 pg rT3). Addition of 50 or
100 nl rT3 * resulted in slightly lower sensitivity.

%B
50

2.0
rTjnrol/l

Figure 11.15.
rT3-standard curves under different incubation times (1,3,5 and 7 days respectively at
4°C). Reaction conditions otherwise as stated under assay procedure.
 39
c. Selection of antiserum dilution. Binding of radioidodinated rT3 to rT3-

i
antiserum no. 3686 in various dilutions is depicted in figure 11.12. More
than 90% of the tracer is specifically bound by excess antiserum. The
optimal antiserum dilution was determined in several experiments. The
I
slope of the standard curve is very similar when employing final antiserum
dilutions 1=100.000 and 1=160.000, although sensitivity is slightly better
in the lower dilution. Further dilution of the antiserum results in a sub-
stantial decrease of sensitivity.
d. Selection of incubation conditions. Several incubation times were tested
(figure 11.15). Sensitivity is highest at the longest incubation time (7 days),
it is equal at incubation times of 5 and 3 days, and is the lowest after one
day of incubation. The slope of the standard curve up to 0.S nmol/1 (up-
per limit of normal range) is steepest at an incubation time of 5 days. Incu-
bation for 5 days at 4°C is therefore preferred.
e. Separation of bound and free hormone fraction. We compared immunol-
ogical precipitation of bound fraction by the double-antibody solid phase
technique (DASP) (Den Hollander and Schuurs 1971) vs. fractional precipi-
tation of bound fraction using polyethylene glycol (Polyaethylenglycolum
6000, Brocacef PO 431) (PEG) (Desbuquois and Aurbach 1971). We
found separation by PEG better and easier to handle due to the firm pellet
obtained than separation by DASP. The PEG technique depends on preci-
pitation of immunoglobulin at a critical concentration of precipitant,
which is largely determined by electrostatic forces, influenced by many
factors such as temperature, pH, protein and salt concentration. Optimal
separation is obtained by the addition of a carrier-7-globulin (e.g. bovine
7-globulin). Instead of bovine 7-globulin we used pig serum which could be
obtained free of charge. Optimal conditions were determined experiment-
ally. In comparison with separation by 12.5% PEG and 0.2% bovine
7-globulin optimal separation in our hands was achieved with 30% PEG
and adding 6 ml pig serum to every 100 ml PEG solution. The final con-
centration of PEG is thus 14% at an incubation volume of 1 ml. Optimal
separation was reached between 10 and 60 minutes after addition of the
PEG solution, but was bad at times shorter than 10 minutes. A separation
time of IS minutes was therefore selected. Pig serum contains iodothyro-
nines which might influence the result of the assay. However, addition
of large amounts of iodothyronines to the pig serum did not interfere with
the measurement of endogenous iodothyronine concentration in the un-
known samples. Obviously, the short incubation with exogenous iodo-
thyronines at a low temperature (4°C) has no effect on the antigen-
antibody equilibrium reached at the end of the first incubation. The sepa-
ration technique was found to be sensitive to changes in temperature,
especially in the rT3 assay: a moderate increase in temperature was asso-
ciated with a decrease in bound hormone. Therefore centrifugation was
 40
carried out in a cooled, temperature-stabilized centrifuge at 4°C.
f. Assay procedure. Reagents are pipetted into 4 ml polystyrene tubes:
x /il buffer up to a final volume of 1 ml
50 Ml blocking agent (300 jug ANS diluted in buffer, freshly prepared) W
SO ml plasma of unknown sample
SO ill iodothyronine-free plasma (in standard curve tubes only)
SO jul standard (in standard curve tubes only, resulting in concentrations of 0.125,
5*
0.25,0.5,0.75 and 1.0 nmol/l rT,) &
50 Ml rT,-antiserum (no. 3686; dilution 1=5000, final dilution 1=100.000)
25 Ml' 2 51-rT3 (containing 10.000 c.p.m. and 0.003125 pmol = 2 pg)
IK
After mixing, the tubes are incubated for 5 days at 4°C. Separation of
bound and free hormone is performed by addition of 1 ml of polyethylene
glycol solution (30 gr. PEG in a final volume of 100 ml buffer to which is
added 6 ml pig's serum). After a separation time of IS minutes at 4°C, the
antibody-bound hormone is collected by centrifugating for IS minutes at
4°C and sucking off the supernatant. The radioactivity in the pellet is
counted in a gamma-scintillation-counter. Each sample is assayed in tripli-
cate. The percent bound hormone is corrected for nonspecific binding by
simultaneously assaying samples in the absence of antiserum.

3a.3. Results.
a. Sensitivity and precision. The precision of the standard curve was deter-
mined by the assay of each standard point in tenfold in one experiment.
The results are given in table II.7 and figure 11.16. Sensitivity is 0.00125
pmol rT 3 /tube, or 0.025 nmol/1 plasma. The detection limit of the assay
is rounded off at 0.03 nmol/1 plasma. There is discrimination between the

I
I
I

0 OSS 025 050 075 1.0

rT] nmol/l

Figure 11.16.
Sensitivity and precision of the rT, standard curve (the mean ± 2SD of the standard
points, each assayed in tenfold, is depicted).
 41
Table 11.7.
Precision of the rT3 standard curve (each point assayed in tenfold).
standard point percentage bound coefficient of variation
(pmoIrT,/tube) mean SD (§B x 100%)

0 50.8 0.99 1.95

0.00625 42.0 0.63 1.50
0.0125 35.8 0.43 1.20
0.025 28.1 0.64 2.28
0.0375 23.3 0.45 1.91
0.05 20.2 0.73 3.61

last two points of the standard curve, and therefore only plasma samples
with a rT3 concentration of > 1.0 nmol/1 have to be reassayed at a higher
dilution. The precision of measuring rT3 levels in unknown plasma samples
is presented in table II .8.
b. Specificity. Non-specific adsorption in the assay system varies between 3
and 7%. There is no difference in non-specific adsorption between the
controls of the standard curve and the controls of unknown plasma
samples: 4/74 ± 0.36% (n=27) and 4.50 ± 0.43% (n=116) respectively
(values as mean ± SD). Therefore, correction for non-specific adsorption
in unknown plasma samples is performed by applying the percentage non-
specific adsorption observed in the standard curve of the assay. No obvious
deviation from parallelism between the standard curve and dilution curves
of several unknown plasmas was noted (figure 11.17). Cross-reactivity to
various related iodothyronines and iodotyrosines in the rT3 assay is
presented in table II.9. Cross-reactivity in these experiments is determined
by comparing the concentrations of rT3 and of the cross-reacting agent (x)
which both displace 50% of the 12S I-rT 3 tracer in the rT3 assay. The
[rT 3 jatl/2B c
percentage cross-reactivity is thus xl00%.
[X] at 1/2 B o
Table 11.8.
Intra-assay variation of unknown plasma samples in the rT3 radioimmunoassay (each
sample assayed in tenfold).
unknown plasma rT3 nmol/1 coefficient of variation
mean SD (^P x 100%)
A 0.11 0.027 24.5
B 0.27 0.040 14.8
C 0.30 0.018 6.0
D 0.41 0.028 6.8
 42
KB
40

ft

0.50 0.75 tO 1.16 1,4 1.2 1.1

tT 3 ranol/l dilution unknown plasma

Figure 11.17.
Parallelism between standard curve and dilution curves of unknown plasma samples in
therT 3 radioimmunoassay.

Since on a molar base plasma levels of T4 are about 460 times higher than
of rT 3 , cross-reactivity of T 4 in the rT3 assay is most important. As can be
seen in figure n.18, concentrations of T 4 exceeding those of rT3 by more
than 3 orders of magnitude are needed to compete with 12S I-rT 3 to the
same extent. Cross-reactivity to T 4 in the rT3 assay as determined in these
experiments, varied between 0.03 and 0.04%, and was the same using dif-
Tabkn.9.
Cross-reactivity of thyroid honnone analogs in the rT3 radioimmunoassay (AS 3686, FD
1=100.000) (cross-reactivity detennined at 50% displacement level of rT, tracer, ex-
pressed on a molar base; cross-reactivity to human thyroglobulin, obtained by courtesy
of Dr. J.J.M. de Vijlder, is expressed by weight).
thyroid hormone analog abbreviation % cross-reactivity

3,3',5' - triiodo - L - thyronine 100

3,5,3',5' - tetraiodo - L - thyronine T« 0.03

3,5,3\5' - tetraiodo - D - thyronine D-T4 0.03
3.5.3' - triiodo - L - thyronine T, < 0.003
3,5 - diiodo - L - thyronine 3,5-T, < 0.003
3,3' - diiodo - L - thyronine 3,3'-^ 0.65
3',5' - diiodo - L - thyronine 3',S'-Tt 1.33
3 - monoiodo - L - thyronine 3-T, < 0.003
3' - monoiodo - L - thyronine 3'-T, 0.06
L - thyronine To < 0.003
3,5,3',5' -tetraiodo -L-thyroaceticacid TETRAC 0.006
3,5.3' -triiodo -L-thyroaceticarid TRIAC < 0.003
3,5 - diiodo - L - tyrosine D1T < 0.003
3 - monoiodo - L - tyrosine MIT < 0.003
thyroglobulin Tg 0.0005
 43
%B
SO

40

30

20

10

0.125 0.25 050 1 25 10 20 50 100 200 500 1000 2000

iodolhyronine nmol/l

Cross-reactivity of T 4 , T 3 and 3,3'-T2 in therT, radioimmunoassay.

ferent batches of T4 (T4 obtained from Henning Berlin GmbH no.

614200, and from Sigma - no. T-2376). Cross-reactivity to T 4 in the rT3
assay was also evaluated by adding increasing amounts of T 4 to four differ-
ent plasma samples (table 11.10). Measured rT3 plasma concentrations are

Table 11.10.

Effect of exogenously added T 4 on measured rT3 concentrations in four different

plasma samples.

mean in- %cross

crease in reactivity
measured ofadded
added T4 measured rT3 (nmol/l) rT3 (nmol/l) T4 inrT3
(nmol/l) plasma E plasma F plasma G plasma H assay

0 <0.03 0.27 0.31 0.39

2.5 <0.03 0.27 0.31 0.39 0 0
5 <0.03 0.27 0.31 0.39 0 0
10 <0.03 0.27 0.31 0.39 0 0
20 <0.03 0.30 0.31 0.39 0.0075 0.0375
50 <0.03 0.33 0.34 0.43 0.03 0.06
100 <0.03 0.37 0.37 0.49 0.07 0.07
200 0.125 0.44 0.42 0.56 0.14 0.07
500 0.25 0.62 0.58 0.80 0.32 0.06
 44
Table 11.11.
Expected and found concentrations of rT3 (nmol/1) in recovery experiments with four
different plasma samples.
added rT3 (nmol/l)
-I: 0.125 0.25 0.50

IV plasma K: expected : - ^ 0.20 0.325 0.575

;
found 0.075 0.20 ; 0.37 0.60
% recovery 100 114 104

plasma L: expected 0.375 0.50 0.75

found 0.25 0.405 0.52 0.90
% recovery 108 104 120

plasma M: expected 0.375 0.50 0.75

found 0.25 0.41 0.57 0.89
% recovery 109 114 119

plasma N: expected 0.40 0.525 0.775

found 0.275 0.395 0.50 0.845
% recovery 99 95 109

consistently higher at additions of 50 nmol/l T 4 and more, and at these

levels exogenously added T 4 cross-reacts for 0.06-0.07% in the rT 3 assay.
The quantitative specificity of the rT 3 assay as judged from recovery
experiments, is presented in table 11.11. The mean percentage recovery
of exogenously added rT 3 hi these twelve experiments is 108%.
c. Quality control The coefficient of inter-assay variation is about 12%
(table H.12).

Table 11.12.
Inter-assay variation of plasma pools in the rT, radioimmunoassay (n= number of assays).
plasma pool rT3 nmol/l coefficient of variation 1
mean SD ($P x 100%)
p 18 0.176 0.026 14.8
Q 19 0.276 0.032 11.6
R 18 0.336 0.040 11.9
S 18 0.419 0.041 9.8
 45
3a.4. Discussion.

The rT3 radioimmunoassay as performed in our laboratory, is very sensitive

(see table 11.14 for comparison with other assays reported in literature). The
low detection limit of the assay has been made possible by the use of a high
specific activity tracer and of an antiserum with high avidity.
Some comments should be made on the preparation o f ' 2s I-rT 3 . Firstly,
from the specific activity obtained in our radioiodination experiments one 11
can conclude that the rT3 tracer is produced mainly by monoradioiodination
\
(i.e. addition of one radioactive iodine atom) rather than by diradioiodination
(i.e. exchange and addition of iodine atoms). This is in accordance with the r
experience that radioiodination of iodothyronines is much more likely to oc-
cur by addition than by exchange of iodine atoms (Kochupillai and Yalow
1978; Nakamura et al. 1978). The rT3 tracer prepared from iodination of
3,3'-T2 is also more stable than radiolabeled rT3 prepared from iodination of
3-Tj, since monoradioiodinated radioiodothyronines are more stable than
diradioiodinated radioiodothyronines (Kochupillai and Yalow 1978). The
obtained high specific activity tracer increased the sensitivity of the assay.
Secondly, by applying the reaction mixture of the radioiodination of 3,3'-
T2 on a large Sephadex G-25 fine column (see figure 11.11) an additional
fourth peak was detected, eluting exactly in the volume of 1 2 S I-T 3 . In this
respect it is of interest that Kochupillai and Yalow (1978) observed an
unidentified shoulder on the rT3 peak, and that Ratcliffe et al (1976) also
detected four peaks, the unidentified third peak eluting at approximately the

Table 11.13.
Substrates and products of radioiodination of thyronines.

substrates radioiodothyronine products formed:

by exchange by addition
one l i 5 I atom two i a s I atoms

T,

3,3'-!, 3,3'-Ta

3,5-T4
3-T, 3,3'-!,
3'-T, 3'-T, 3',5'-Ta
3'-T, 3',5'-T,
v.Sh j-

Table II-14
Main features of iT, radioimmunoassays as reported in the literature

REFERENCE TRACER ANTISERUM STANDARD' rTj-TBP INCUBATION SENSI- % CROSS-REACTI-

F.D. NEUTRAL- TIVITY VITY1
specact. pg added IZATION incubation separation nmol/1 T« 3,3'-^
MCi/Mg in assay B/F
Chopra 1974 400-500 30-40 1=250 Meltzer ethanol 24 hrs. 4°C DA 0.15 0.07 8.1
extraction
Meinhold 1975 ±3300 6 1=18.000 Henning ethanol 2 hrs. room temp. DA 0.03 0.025-0.1 1.0
extraction
Nicod 1976 1=6.400 Cahnmann ANS 300Mg 2 hrs. -40°C QAE 0.15 0.07 0.5
16 hrs.-room temp. Sephadex
2 days-4° C A-25
Ratdiffe 1976 6 1=40.000 Henning ANS 250/ig 16 hrs. 4o C DA 0.05 0.14 0.42
Griffiths 1976 1=13.500 Henning ANS 175pg 2 hrs. room temp. DA 0.08 0.08
16-20 hrs. 4°C
Hüfner 1976 1=25.000 Henning ANS 150Mg 20 hrs. 22° C DASP 0.09 ±0.06 <0.01
Visser 1977 ±2700 4 1=100.000 Henning ANS 500ng 2 days 10°C DA 0.03 0.035 0.57
Burrows 1977 1=315.000 Henning 0.12 0.02
Burman 1977 350-400 10 1=5.000 Cahnmann ANS 300/ig 18 hrs. room temp. (NH 4 ) a S0 4 0.15 <0.01 0.08
Kaplan 1977 400-500 variable Meltzer ANS 500Mg 16 hrs. 4°C DA 0.06 0.05 0.4
Gavin 1977 ±500 1=10.000 Jorgensen ANS DA 0.08 0.07 3.1
Laurberg 1977 3200 2 1=1.450 Henning ANS 75/ig 18 hrs. room temp. DE cellulose 0.024 0.075 0.23
Kochupillai 1978 3500 1 1=3.000 Meltzer ANS 200Mg 16 hrs. charcoal 0.015
Faber 1978 1=800 Henning Sephadex 16 hrs. room temp. Sephadex 0.07 0.04-0.07 2.3
G-2S fine P-25 fine
Mathur 1979 2 Henning ANS 250*ig 16 hrs. 4°C charcoal 0.015 "insignificant"
Wiersinga 1979 ±3150 2 1=100.000 Henning ANS 300jig 5days4°C PEG 0.03 0.035 0.65

iJTSiS.' -J WkÄJUÄSi-iiAHi
' TV'

REFERENCE NORMAL VALUES (nmol/l) HYPERTHYROIDISM HYPOTHYROIDISM

mean ± SD range n mean ± SD range mean ± SD range

Chopra 1974 0.62 ±0.16 0.42-0.9S 27 1.58 ± 0.75 0.83 - 3.54 22 0.29 ± 0.14 <0.15- 0.54 12

Meinhold 1975 0.28 ± 0.18 0.03-0.71 45 0.801 0.53 0.24 - 1.88 28 0.06 <0.03-0.19 15

Nicod 1976 0.69 ± 0.31 0.38-0.99 83 2.40 1.17-3.97 11 <0.15-0.61 12

Ratcliffe 1976 0.27 ± 0.06 0.15-0.42 44 1.26 ±0.76 0.41-4.66 58 0.10 ±0.04 0.05-0.25 31
0.47 ±0.25 0.21-1.18 153 <0.05 23
Griffiths 1976 0.68 ±0.16 0.43-0.90 67 1.18 ±0.49 0.71-2.50 18 0.19 ±0.07 <0.06-0.33 12
0.42 ±0.16 0.16-0.74 16s
Hufner 1976 0.31 ± 0.14 <0.09 - 0.69 60 ±1.00 ±0.46 ±0.30-2.80 34 most<0.09 14
Visser 1977 0.22
Burrows
Burman
1977
1977 0.92 ±0.18 0.63-1.37 21 2.60 ± 0.26 0.91 - 3.96 19 0.29 <0.15-0.85 11
1
Kaplan 1977 0.35 ± 0.12 106 1.39 ± 0.75 22 0.22 ± 0.08 21
0.55 ± 0.20 53
Gavin 1977 0.37-0.65 6
Laurberg 1977 0.37 ± 0.099 0.18 - 0.58 32 1.35 i 0.37 0.81 - 1.98 17 <0.024 - 0.046 10
Kochupillai 1978
Pabcr 1978 0.74 ±0.14 0.45-1.09 58 2.48 ±0.77 1.60-4.74 30 0.37 ±0.06 0.25-0.42 12
0.86 0.82 - 0.88
Mathur 1979 0.29 ± 0.07 0.17-0.42 40 1.61 ±0.99 0.40-5.67 48 0.07 ±0.06 0.015-0.27 35
<0.015 5
Wiersinga 1979 0.24 ± 0.07 0.11 - 0.44 63 0.81 ± 0.54 0.26 - 4.05 55 0.06 ± 0.05 <0.03 - 0.17 33
0.42 ±0.16 0.22-0.69 II 3
1
standard: Dr. Meltzer = DL-rT,, others L-rT,
2
on a molar base
J
Tj-toxicosis

sfetiii-V'SsSf'-*«i;irfii*i4itLcSit'ii"--''^"i1"'.1* V-t Jn*»es.*"«i*J''5Vt*i' » ^C ^ .J. -.

:
'^r'^~Tm*^7^^
& same volume as in our experiment. The radioiodothyronines which can be '£&
fc
produced by radioiodination of the various thyronines as substrate, are listed %/;
in table 11.13. It must be noted that iodination in the inner ring does not ||
occur under these circumstances, probably caused by the steric structure of fj^
the thyronine molecule. It can be speculated that the unknown peak consists « ||
of labeled 3,5,3'-T3, which is formed by addition of radioiodine to 3,5-T2 fe
present as contaminant in the 3,3'-T2 batch, since a) the elution volume is |-
that of 12 S I-T 3 b) the chance of radioiodination by addition is greater than
by exchange, which favours 3,5-T2 over 3,5,3'-T3 as the contaminant present
as substrate for T 3 * (according to Henning Berlin GmbH the 3,3'-T2 prepara-
tion is pure for > 99% and contains < 0.5% T 3 ) c) it is improbable that the
identity is 12S I-3',5'-T 2 (formed by addition from T o or 3'-Ti, or by ex-
change from 3',5'-T2, present as contaminants in the 3,3'-T2 batch) as 3',5'-
T 2 elutes earlier than T 3 .
The Henning L-rT3 preparation is used as standard in the rT3 assay. rT3
standards from various sources have been shown to behave differently in the
same rT3 assay (Premachandra 1978). The slopes of the standard curves
differed considerably, and employment of the Henning standard resulted in
the steepest slope. The concentration of rT3 in one particular human serum
as measured with the Henning standard was 0.31 nmol/1, and 0.65 nmol/1
when Jorgensen's L-rT3 standard was used. The DL-rT3 preparation of Dr.
Meltzer has approximately half of the immunological potency as Henning's \\
rT3 (Burrows et al 1977). Premachandra argues that differences in tech-
niques for the synthesis and purification of rT 3 , and not measurable T 3 or T 4
contamination of rT3 constitutes to the different results obtained with the
various rT3 standards, since these standards even in high concentration did
not bind to T 3 - or T 4 -antisera in the T 3 - and T 4 -RIA's. However this may be,
it is clear that different rT3 standards can result in a different range for nor-
mal values in the rT3 assay.
All authors performing the rT3 assay in unextracted serum or plasma, em-
ploy ANS to dissociate the binding of rT3 to TBP (see table 11.14). Omittance
of ANS results in a substantial decrease of specific binding of the rT3 tracer
(figure 11.14), and is in itself proof of the binding of rT3 to plasma proteins.
The concentrations of ANS chosen will completely block the rT3 protein
binding in most cases, but it cannot be excluded that incomplete blocking
may occur at very high TBP concentrations.
Separation of the bound and free hormone fractions in our rT3 assay is
performed by polyethylene glycol, but judging from the literature other sepa-
ration techniques work equally well (table 11.14).
Cross-reactivity to T 4 in our assay was 0.03-0.04% when determined by
50% displacement of the rT3 tracer, and 0.06-0.07% when determined by the
effect of exogenously added T 4 on measured rT3 concentration in a plasma
sample. The parallelism between the 3,3'-T2 and T 4 curves and the rT3
V. 49
standard curve (figure 11.18) may indicate contamination with rT 3 of the
3,3'-T2 and T 4 preparations rather than real cross-reactivity of the antiserum.
1
The percentage cross-reactivity to T 4 is rather low when compared to the
figures in the literature (table 11.14), although some authors report still lower
cross-reactivity. Burrows et al (1977) succeeded in obtaining a T 4 -cross-
reactivity of 0.02% by preabsorption of the antiserum with T 4 , albeit at the
I
cost of a loss in sensitivity. In principle this procedure may also be applied to
the antiserum 3686, since due to its very good avidity some loss in sensitivity
may be acceptable if cross-reactivity substantially decreases. Other authors
have sought to solve the problem of T4-crossreactivity in the rT3 assay by
correction of the measured rT 3 concentrations forT4-crossreactivity. Correc-
tion for cross-reactivity however is a complicated matter which is not per-
mitted unless the characteristics of the antibody are very meticulously tested
(Mathur et al 1979), and we do not apply any correction for cross-reactivity.
In practice, it is possible that the presence of T 4 in a concentration of 100
nmol/1 in a plasma sample is measured as 0.03 nmol/1 in the rT3 assay.
Changes in T 4 concentration of SO nmol/1 and more may interfere with the
- rT 3 measurements, and must be taken into account when interpreting the rT 3
levels. Crossreactivity of 3,3'-T2 is greater than of T 4 in the rT 3 assay, but
this is of no practical importance since the 3,3'-T2 concentration in normal
i; human serum is approximately 0.02 nmol/1 (Visser et al 1978). Even the
highest reported normal values for 3,3'-T2 of 0.3 nmol/1 results in rT 3 values
ten times less than the detection limit of the assay. For similar reasons, the
crossreactivity to 3',5'-T2 in the rT 3 assay can be neglected.
The described assay for rT 3 further meets the requirements of precision,
reproducibility, and quantitative specificity (parallelism and recovery). The
reported values for the normal range of plasma rT 3 vary considerably (see
table 11.14) and this may be due to different technical aspects of the assays
(especially differences in sensitivity and specificity with regards to T 4 ), to
different rT 3 standards and to different ways of correcting for T 4 -cross-
reactivity.

3a.5. References.

i Blasi F, De Masi R V. Adsorption properties of iodotyrosines and their derivatives on

8 Sephadex. J Chromatogr 1967; 28: 33-6.
Burger A, Ingbar S H, Labelling of thyroid hormones and their derivatives. Endocrinology
1974:94:1189-92.
Burman K D, Dimond R C, Wright F D, Earll J M, Bruton J, Wartofsky L. A radio-
immunoassay for 3,3',5'-L-triiodothyronine (reverse T 3 ): assessment of thyroid
gland content and serum measurements in conditions of normal and altered thyroidal
economy and following administration of thyrotropin releasing hormone (TRH) and
thyrotropin (TSH). J Clin Endocrinol Metab 1977; 44: 660-72.
Burrows A W, Cooper E, Shakespear R A, Aickin C M, Fraser S, Hesch R D, Burke C W.
Low serum L-T3 levels in the elderly sick: protein binding, thyroid and pituitary
1
h 50 . $>"
f. i , lv
|>, responsiveness, and reverse T 3 concentrations. Clin Endocrinol 1977; 7: 289-300. *$•*.
'('2 Chopra I J, A radioimmunoassay for measurement of thyroxine in unextracted scrum. ', -££
Sf J Clin Endocrinol Metab 1972; 34:938-47. ; *£
?£ Chopra I J, A radioimmunoassay for measurement of 3,3'.5'-triiodothyronine (reverse jf;,
$ T s ) . JClin Invest 1974; 5 4 : 583-92. i §
§ Desbuquois B, Aurbach G D . Use o f polyethylene glycol to separate free and antibody- « p.
\~ bound peptide hormones in radioimmunoassays. J Clin Endocrinol Metab 1971; |;';"
ii 33:732-8. : $
£' Faber J, Friis T, Kirkegaard C, Siersbaek-Nielsen K. Radioimmunoassay of 3,3',5'- K
I} triiodothyronine (reverse T 3 ) on small reusable Sephadex columns. Acta Endocrinol J
SJ 1978; 87: 313-9.
Gavin L, Castle J, McMahon F, Martin P, Hammond M, Cavalicri R R. Extrathyroidal
conversion of thyroxine to 3,3\5'-triiodothyronine (reverse T 3 ) and to 3,5,3'-
triiodothyronine (T 3 ) in humans. J Clin Endocrinol Metab 1977; 44: 733-42.
Griffiths R S, Black E G, Hoffenberg R. Measurement of serum 3,3',5'-(reverse) T 3 , '
with comments on its derivation. Clin Endocrinol 1976; 5: 679-85. |
;
Hollander F C den, Schuurs A H W H . Discussion. In: Kirkham K E, Hunter W M, eds. ;
Radioimmunoassay methods. Churchill Livingstone, Edinburgh: 1971: 419-22. • |-
Hiifner M, Hesch R D. A comparison of different compounds for TBG-blocking used in .; j \
radioimmunoassay for triiodothyronine. Clin Chim Acta 1973; 44: 101-7. (
Hiifner M, Grussendorf M. Radioimmunoassay for 3.3',5'-triiodothyronine (reverse T 3 , ]
r-T,) in unextracted human serum. Clin Chim Acta 1976; 69: 497-504. =
Kaplan M M, Schimmel M, Utiger R D. Changes in serum 3,3',5'-triiodothyronine (rever- •
se T 3 ) concentrations with altered thyroid hormone secretion and metabolism. J Clin
Endocrinol Metab 1977; 447-56.
Kochupillai N, Yalow R S. Preparation, purification, and stability of high specific
activity' "Mabeled thyronines. Endocrinology 1978; 102: 128-35. •
Kodding R, Hesch R D, Miihlen A von zur. A radioimmunoassay for reverse triiodo-
thyronine. Acta Endocrinol 1976; Suppl. 102:47-9.
Larsen P R. Direct immunoassay, of triiodothyronine in human scrum. JClin Invest 1972;
51:1939-49.
Laurberg P, Weeke J. Radioimmunological determination of reverse triiodothyronine in
unextracted serum and serum dialysates. Scand J Clin Lab Invest 1977; 37: 735-9. .;
Lissitzky S, Bismuth J, Rolland M. Separation des composes iodes du serum et de la ;
thyroTde par filtration sur gel de dextrane (Sephadex). Clin Chem Acta 1962; 7: i
183-9. •
Mathur H, Ekins R P, Brown B L, Malan P G, Kurtz A B. Correction for the presence ]
of cross-reactants in saturation assays: application to thyroxine cross-reactivity in
3,3',5'-(reverse)-triiodothyronine radioimmunoassay. Clin Chim Acta 1979; 91: •
317-27. i
| Meinhold H, Wenzel KW, Schiirnbrand P. Radioimmunoassay of 3,3',5'-triiodo-L-thy-
:
!f. ronine (reverse T 3 ) in human serum and its application in different thyroid states. .
|| JClin ChemClinBiochem 1975; 13;571-4. ;
% Mitsuma T, Gershengorn M, Colucci J, Hollander CS. Radioimmunoassay of triiodo- j
% thyronine in unextracted human serum. J Gin Endocrinol Metab 1971; 33: 364-7.
\} Mougey E H, Mason J W. Separation of some iodoamino acids and iodide by gel filtra- i
;• tion. Anal Biochem 1963; 6:223-33. ..
| Nakamura Y, Chopra I J, Solomon D H. Preparation of high-specific-activity radioactive |
j iodothyronines and their analogues. J Nud Med 1977; 18:1112-5. \
^ Nicod P, Burger A, Staeheli V, Valloton M B. A radioimmunoassay for 3,3',5'-triiodo- ]
., L-thyronine in unextracted serum: method and clinical results. J Clin Endocrinol i
Metab 1976; 42:823-9. i
 -.•M-J--

t3 51
Premachandra B N, Radioimmunoassay of reverse triiodothyronine. J Clin Endocrinol
Metab 1978; 47: 746-750.
Ratcliffe W A, Marshall J, Ratcliffe J G. The radioimmunoassay of 3,3',5'-triiodothy-
ronine (reverse T 3 ) in unextracted human serum. Gin Endocrinol 1976; S: 6 3 1 4 1 .
Sterling K, Milch P O. Thermal inactivation of thyroxine-binding globulin for direct
radioimmunoassay of triiodothyronine in serum. J Clin Endocrinol Metab 1974;
38: 866-75.
Visser T J, Docter R, Hennemann G. Radiounmunoassay of reverse triiodothyronine.
PY
J Endocrinol 1977; 73: 395-6.
Visser T J, Krieger-Quist L M, Doctor R, Hennemann G. Radioimmunoassay of 3,3'-di-
iodothyronine in unextiacted serum: the effect of endogenous triiodothyronine.
J Endocrinol 1978; 79: 357-62.

V
 52
II.3b. Triiodothyronine(7\).

Although triiodothyronine was identified in human plasma by Gross and

Pitt-Rivers as long ago as 1952, it was, due to the cumbersone techniques
needed for measurement of T 3 , not until the late sixties that new interest
I
in the fate of T 3 was awakened by the introduction of less difficult techni-
ques. T 3 was measured by a competitive protein binding technique, after
extraction of T 3 from human serum and separation of T 3 andT 4 by chroma-
tography (Nauman etal 1967,Sterling et al 1969). However, these techniques
also were subject to severe artifacts (Fisher and Dussault 1971; Larsen 1971).
The development of radioimmunoassays for the measurement of T 3 begins in
the early seventies, after the report of Ekins and co-workers (Brown et al
1970) that specific T 3 antibodies could be obtained by immunization of rab-
bits with T3-protein conjugates. At present the T3-RIA is the accepted meth-
od for measurement of T 3 in human plasma. We applied the technique of
Larsen (1972), who employs sodium salicylate as TBP blocking agent.
£- I

3b. 1. Ingredients.
a. Buffer-solution. The composition of the buffer is: sodium barbital 0.075
M, merthiolate 10" 4 M, bovine serum albumin 0.25% in distilled water,
pH=8.6bylMHCl.
b. T3Standard. 3,5,3'-triiodo-L-thyronine (free acid) was purchased from
Sigma, St. Louis, Mo, USA. Standard preparations were made by dissolving
the agent in 0.01 M NaOH/propylene-glycol (1:2), and further dilution in
buffer. Storage was at -20°C.
c. T3-Antiserum. Four rabbits ("Vlaamse reuzen") were immunized against a
conjugate of T 3 and bovine thyroglobulin (Sigma) as described by Chopra
(1972). After three months all rabbits had developed antisera to T 3 of
good avidity.
d. T3-Tracer. 1 2 S I-T 3 is purchased from Abbott Laboratories, North Chica-
go, Illinois, USA. The specific activity of this preparation is ± 450 juCi//ig.
Before use the tracer is purified by Sephadex G-25 chromatography
(column size 50x1 cm; elution buffer 0.01 M NaOH in distilled water,
pH=12). The elution pattern is similar to the profile shown in figure 11.11
(middle part). Storage of the tracer, diluted in buffer to ± 10.000 c.p.m./
50/il,isat-20°C.

3b.2. Assay procedure.

a. Milieu. Iodothyronine-free plasma prepared by the resin-technique (see
section 3a.2a.) is employed in the tubes of the standard curve. Three
agents were compared with respect to their effect on dissociating T 3 from
its binding to TBP: merthiolate (Thiomersal, BDH Chemicals Ltd, no.
30416; 250 jig/tube), salicylate (sodium salicylate cryst. GR, Merck, Art.
 53
6601; 3000 /ng/tube), and ANS (8-anilino-l-naphtalene sulfonic acid, ffci
Sigma, no. A-312S; 125 jug/tube). T 3 concentrations in 23 human plasma
samples were measured, the samples being assayed separately with each of
the three agents. The results (values as mean ± SD) are: 2.60 ± 1.05 nmol/1
(merthiolate), 2.23 ± 0.99 nmol/1 (salicylate), 2.56 ± 1.03 nmol/1 (ANS).
There is no difference in T 3 plasma concentration when merthiolate or
S
ANS is used, but salicylate addition results in lower values as compared to
$ addition of merthiolate (p<0.01) and of ANS (p<0.001) (paired t-test).
b. Tracer concentration. Approximately 85 pg T 3 * per tube (± 10.000
c.p jn./50 Ml) is added.
c. Antiserum dilution. Out of the four antisera obtained, antiserum 137
demonstrated the highest binding of T 3 -tracer, and this antiserum was
used exclusively. The tracer is bound for more than 90% by excess anti-
serum (figure 11.12, middle part). The optimal final dilution of this anti-
serum is 1=140.000.
d. Incubation conditions. The test tubes are incubated for 18-24 hours at
4°C.
e. Separation of bound and free hormone fraction. Although the dextran-
coated charcoal technique was initially used, we subsequently changed to
polyethylene-glycol, because PEG was easier to handle and less subject to
nonspecific protein interference (for details see under section 3a.2e.). I'
f. Assay procedure. The following reagents are consecutively pipetted in 4 ml %-•

polystyrene tubes:
x Ml buffer up to a final volume of 1 ml
200 Ml buffer containing 3 mg sodium salicylatc
100 Ml standard (containing 0-2 pg T3/jnl) in standard curve tubes only
50 nl iodothyronine-free plasma (in standard curve tubes only)
SO Ml unknown plasma sample
50/il tracer (containing 10.000 c.p.m., ± 85 pgT 3 )
100 Ml AS 137, FD 1=140.000
The tubes are incubated for 18-24 hours at 4°C, whereafter the bound
fraction is separated from the free by polyethylene-glycol (see under sec-
tion 3a.2f.)
Each sample is assayed in triplicate. The percent bound hormone is cor-
rected for non-specific binding by simultaneously assaying samples in the
absence of antiserum.

3b. 3. Results. •1
a. Sensitivity and precision. The detection limit of the assay is 0.1 nmol/1.
The intra-assay variation is 12% at a plasma T 3 concentration of 0.9 nmol/ I
1 and 5% at a level of 2.0 nmol/1. 1
b. Specificity. Cross-reactivity to related compounds is presented in table
11.15. Assuming that the T 4 preparation used in these experiments is not
contaminated with T 3 , the cross-reactivity of T 4 in the T 3 assay is at most

fc
 54
Table III5.
Cross-reactivity of thyroid hormone analogs in the T3 radioimmunoassay (AS 137, FD=
140.000) (see also legend table II.9).
1U
thyroid hormone analog abbreviation % cross-reactivity

3,5,3' - triiodo - L - thyronine T3 100

i§
3,5,3',5' - tetraiodo
- triiodo
- L - thyronine
- L - thyronine
T« <0.1 I
3,3',5' rT3 0.0031
3,5 - diiodo - L - thyronine 3,5-T, 0.18
3,3' - diiodo - L - thyronine 3,3'-Ta 0.63
3\5' - diiodo - L - thyronine 3',5'-T, < 0.0005
3,5,3!,5' - tetraiodo - L - thyroacetic acid TETRAC 0.077
3,5,3' - triiodo - L - thyroacetic acid TRIAC 8.3
thyroglobulin Tg 0.62

0.1%; this means an overestimation of T 3 concentrations with 6% for the

mean normal plasma T 3 level of 1.8 nmol/1.
No obvious deviation from parallelism between the standard curve and
dilutions of unknown plasma samples was observed. The recovery of
exogenously added T 3 in ten experiments, was on average 101% with a
range from 80 to 120%.
c. Quality control Inter-assay variation is 13.7% at a plasma T 3 concentra-
tion of 0.79 nmol/1, 9,6% at 2.08 nmol/1, and 5.6% at 4.42 nmol/1 (sam-
ples assayed in 26 consecutive experiments).

3b.4. Discussion.

Antibodies to T 3 are easily obtained by immunization of rabbits with

thyroglobulin, with thyroid microsomal fraction, or with conjugates of T 3
coupled to serum albumin (Chopra et al 1971; Gharib et al 1971). Thyro-
globulin injected in Freund's adjuvant was the most effective antigen in these
studies; the antisera thus produced have sometimes a higher titer of "T3-
specific" binding sites, sometimes the avidity to T 4 is the highest (Chopra et
al 1971). Our experience is similar: immunization of rabbits with Tg resulted
in antisera with highest avidity to T4 which are used in the T 4 radioimmuno-
assay (see figure 11.12), but also in antisera with highest avidity to T 3 . How-
f
ever, in the T 3 assay we preferred antisera obtained by immunization with T 3
I coupled to Tg (Chopra 1972), since these antisera demonstrated lower cross-
k reactivity to T 4 . In general, all these antibodies seem to recognize the thy-
I
ronine structure and the number and location of the iodine atoms; the iodine
atom attached at the 5' location is probably a major antigenic determinant.
 55
The specific activity of the T 3 tracer employed in most T 3 assays is be-
tween 70 and 650 MCi/jug/tube, table 11.16). This is far below the theoretical
maximum of 3500 juCi/^g. High specific activity tracers of about 3000 juCi/
jug can easily be produced by addition of one radioiodine atom to 3,5-T2, by
which the sensitivity of the T 3 assay increases to 0.0015 nmol/1 (Weeke and s
Orskov 1973). The normal range of plasma T 3 concentration is approximately
1.0-2.5 nmol/1, and consequently high specific activity tracers associated with
great sensitivity are not needed. There is good agreement in the reported values
of plasma T 3 between measurements with low and high specific activity tracers k
(Weeke and Orskov 1975). Tracers of high specific activity are applied in the
direct determination of free triiodothyronine concentration in serum, which is
in the order of 0.009 nmol/1 (Weeke and Orskov 1975).
Much literature is available on the compounds used for the displacement
of T 3 from its binding to the serum proteins. Anilino-naphtalene-sulfonic
acid (ANS) is reported to be most effective as blocking agent, although it also
decreases the binding of the tracer to the antibody; this effect is partially
abolished by addition of plasma (Hufner and Hesch 1973). Salicylate de-
monstrated similar effects, but the decrease in binding could be completely
avoided by addition of plasma. As demonstrated by Larsen (1972), salicylate
is more effective in displacing T 3 than T 4 from the binding proteins; theo-
retically this is of advantage, since cross-reactivity to T 4 in the T 3 assay there-
by diminishes. We applied salicylate as blocking agent. Merthiolate, a deriv-
ative of salicylate, is less frequently used in T 3 assays. Hufner and Hesch
(1973) recommend the use of merthiolate as blocking agent, since it would
not influence the antigen-antibody reaction. However, in later studies merthi-
olate was also found to inhibit the antibody binding reaction (Sterling and
Milch 1974; Kanagasabapathy and Wellby 1974).
Incubation conditions were not tested in detail for the T 3 assay in our
laboratory. Possibly, a much shorter incubation for a few hours at 37°C
may also be sufficient to reach equilibrium (Standefer; Lenz et al 1976).
Polyethylene glycol was chosen as separation method because of its low cost,
reliability and rapidity, but other separation procedures can also be used
(table 11.16).
Cross-reactivity to T 4 in the T 3 assay is important since plasma T 4 concen-
trations are about 60 times higher than of T 3 (on a molar base). Most assays
demonstrate a T4-reactivity of 0.1% or less (table 11.16). The question is, if
this represents real cross-reactivity or T 3 contamination of the T 4 preparation
used. The latter possibility is suggested by reports that T4-reactivity in a
particular T 3 assay varies with different T 4 batches (Patel and Burger 1973;
Twomey and Sweat 1975; Seth et al 1976).
it The T 3 radioimmunoassay as performed in our laboratory is sensitive and
precise, and meets the requirements for qualitative and quantitative specif-
icity and quality control. The normal values obtained for the plasma T 3 con-
Main features of T, ladioimmunoassays, as repotted in the literature

REFERENCE TRACER ANT1SERUM T,TBP INCUBATION SENSI- % CROSS-REAC-

NEUTRAL- TIVITY TIVITY'
IZATION
specact PgT 3 immuni- FD incubation separation nmol/l
MCI/Mg added zation B/F

Gharib 1971, 90-120 SO T 3 -HSA 1=1000/ merthiolate 0.02% s p r e 2 4 h r s . 4°C DA 0.77 0.1-0.4
1972 1=4000 8 hrs. 4°C
Mitsuma 1971 70-90 30 T 3 -BSA 1=400 0.2 Mg tetra- 24 hrs. 4°C charcoal 0.20 0.02
chlorothyronine
Larsen 1972 300-500 < 3 0 T 3 -BSA 1=106.000 salicylate pre 2-5 days 4°C charcoal 0.10 0.1
7 mg/tube 2-3 days 4° C
Chopra 1972 90-100 100-150 T 3 -Tg 1=4000 ANS 250 Mg 24 hrs. 4°C DA 0.18-0.31 0.06
Surks 1972 70-90 75 T 3 -BSA 1=8000 Sephadex 18-20 hrs charcoal 0.09 <0.1
4°C
Lieblich 1972 50 40-80 T 3 -BSA 1=800 DPH 5 0 Mg/tube 48 hrs. 5°C DA 0.39 0.2-1.6
Patel 1973 70-83 40-50 T 3 -BSA 1=125.000 ethanol pre 24 hrs. 4°C DA 0.09-0.22 0.2-1.4
extraction 48 hrs. 4° C
Hüfnet 1973 80-110 40-60 T 3 -BSA 1=20.000 merthiolatc 24 hrs. 4° C charcoal 0.31 0.4-0.6
Sterling 1974 540 T 3 -BSA 1=5000 60°C2hrs. Ihr. 60°C PEG 0.15 "negligible"
18 hrs. 4°C
Meinhold 1974 500 33 T 3 -BSA 1=180.000 ANS 200 Mg 1 hi. room DA ±0.18 0.05
temp.
Twomey 1975 500 50 T 3 -BSA 1=5000 ANS 400 Mg 3 hrs. 37° C PEG 0.39 0.6-1.6
Burman 1975 600 25-50 1=150.000 ANS 250 Mg 1 hr. 37° C resin 0.39 <0.4
Burger 1975 100 T 3 -BSA ANS 80Mg 2 hrs. 40°C QAE 0.2-0.4
48 hrs. 4° C Sephadex
A-25
Seth 1976 651 5 1=15.000 ANS 250 Mg 24 hrs. 4°C antibody- 0.29 0.02
wash solution 3
Stepanas 1977» merthiolate 18 hrs. room resin <0.2
temp.
Wiersinga 1979 450 85 T 3 -Tg 1=140.000 salicylate 18-24 hrs. PEG 0.1 <0.1
3 mg/tube 4°C
 V;. .'t,'" ,.•-:••

REFERENCE NORMALS (ntnol/l) HYPERTHYROIDISM HVPOTHYROIDISM

mean ± SD range mean ± SD tange mean ± SD tange

Gharib 1971, 3.31 ± 0.85 1.85 - 4.80 11.70 ±4.45 5.67-22.68 1.59 ±0.66 0 . 6 2 - 2.51
1972
Mitsuma 1971 2.13 ±0.35 1.48-2.65 82 7.61 ± 4.08 3.82 - 26.18 60 0.95 ±0.14 0 . 6 8 - 1.23 45
10.96 ± 6.41 3.51 - 30.80 404
Laiscn 1972 1.69 ± 0.39 38 8.41 ± 6.81 24 0.60 ± 0.32 25
Chopra 1972 1.74 ± 0.50 0.69 - 3.33 96 7.56 ± 3.57 30 0.62 ±0.41 < 0 . 1 8 - 1.60 12
Sutks 1972 2.25 ± 0.37 1.54 - 3.02 38 10.24 ± 4.95 4.45 - 20.02 22 0.68 ±0.40 0.11 -<1.54 29
Lieblich 1972 2.23 ± 0..?9 79 6.61 ± 2.25 36 1.52 ± 0.37 45
Patel 1973 2.00 1.34-2.73 29<5 6.93 2.71 - 17.71 26 0.65 < 0 . 0 9 - 2.11 24
1.96 1.16 - 2.54 209
Hüfner 1973 1.85 ± 0.46 1.39 - 2.31 15 4.62 - 46.20 15 <0.31 - 0.777
Sterling 1974 2.91 ± 0.46 31 12.91 ± 6.13 14 0.72 ± 0.60 19
Meinhold 1974 1.77 ± 0.37 1.04 - 2.46 68 6.30 ±3.18 2.57 - 23.5 89 «C0.18- 2.63 62
3.69 ± 1.24 2.47- 7.684 764
Twomey 1975 2.17 1.02 - 3.31 49
Buiman 1975 1.91 ± 0.35 1.17 - 2.68 32 5.05 ± 1.74 2.80 - >9.24 23 0.63 ± 0.31 <0.39 - 1.25 17
Burger 1975 2.57 ± 0.59 99 3.39 - 16.02 31 0 - 1.23 10
Seth 1976 1.70 .1.12 - 2.21 52
Stepanas 19771 2.11 ±0.46 1.19 - 3.02 335
Wiersinga 1979 1.85 ± 0.37 1.00 - 2.85 63 4.94 ±2.51 2.50-13.35 55 0.94 ±0.49 0.19- 2.15 33
3.01 ± 0.56 2 . 5 3 - 3.94 II 4

' on a molar base

'T,-RIA kit, The Radiochcmical Centre, Amcrsham
'solid phase RIA with use of antibodies coupled to Scpharosc
4
T,-toxicosis
9
pre = preincubation
 - •._—i--.;

58
centrations are in the same Older as described in the literature, although at
the lower end of the reported values (table 11.16).

3b.5. References.
Brown B L, Ekins R P, Ellis S M, Reith W S. Specific antibodies to T 3 . Nature 1970;
226: 359.
Burger A, Sakaloff:C, Staeheli V, Valloton M B, Ingbar I H, Radioimmunoassays of
3,5,3'-triiodo-L-thyronine with and without a prior extraction step. Acta Endocrinol f
1975; 80: 58-69.
Burman K D, Wright F D, Earll J M, Wartofsky L. Evaluation of a rapid and simple tech-
nique for the radioimmunoassay of triiodothyronine (T 3 ). J Nucl Med 1975; 16:
662-5.
Chopra I J, Nelson J C, Solomon D H, Beall G N. Production of antibodies specifically
binding triiodothyronine and thyroxine. J Clin Endocrinol Metab 1971; 32: 299-
308.
Chopra I J, Ho R S, Lam R. An unproved radioimmunoassay of triiodothyronir. ^ ,<)
serum: its application to clinical and physiological studies. J Lab Clin Med 1971, -; J:
729-39.
Fisher D A, Dussault J H. Contribution of methodological artifacts to the measurement
of T 3 concentration in serum. J Clin Endocrinol Metab 1971; 32: 675-9.
Ghaiib H, Ryan R J, Mayberry W E, Hockert T. Radioimmunoassay for triiodothyronine
(T 3 ): I. Affinity and specificity of the antibody for T 3 . J Clin Endocrinol Metab
1971; 33: 509-16.
Gharib H, Ryan R J, Mayberry W E. Triiodothyronine (T 3 ) radioimmunoassay. A critical
evaluation. Mayo CHnProc 1972; 47: 934-7. —
Gross J, Pitt-Rivers R. The identification of 3,5,3'-L-triiodothyronine in human plasma.
Lancet 1952; 1: 439-41.
Hiifner M, Hesch R D. A comparison of different compounds for TBG-blocking used in
radioimmunoassay for tri-iodothyronine. Clin Chim Acta 1973; 44:101-7.
Hufner M, Hesch R D. Radioimmunoassay for T 3 in human serum. Acta Endocrinol
1973: 72:464-74.
Kanagasabapathy A I, Wellby A S. The use of merthiolate for TBG-blocking in the
radioimmunoassay of triiodothyronine. Clin Chim Acta 1974; 55: 267-71.
Larsen P R. Technical aspects of the estimation of triiodothyronine in human serum:
evidence of conversion of thyroxine to triiodothyronine during assay. Metabolism
1971; 20: 609-24.
Larsen P R. Direct immunoassay of triiodothyronine in human serum. J Clin Invest
1972:51:1939-49.
Lieblich I, Utiger R D. Triiodothyronine radioimmunoassay. J Clin Invest 1972; 51:
157-66.
Meinhold H, Wenzel K W. Studies on the direct radioimmunological determination of
triiodothyronine in serum. J Clin Chem Gin Biochem 1974; 12:477-86.
Mitsuma T, Nihei N, Gershengorn M C, Hollander C S. Serum triiodothyronine: meas-
urements in human serum by radioimmunoassay with corroboration by gas-liquid
chromatography. J Clin Invest 1971; 50: 2679-88.
Nauman J A, Nauman A, Werner S C. Total and free triiodothyronine in human serum.
J Clin Invest 1967; 46:1346-55.
Patel Y C, Burger H G. A simplified radioimmunoassay for triiodothyronine. J Clin
Endocrinol Metab 1973; 36:187-190.
Seth J, Toft A D, Irvine W J. Simple solid-phase radioimmunoassays for total tri-iodo-
 59
thyroninc and thyroxine in serum, and their clinical evaluation. Clin Chim Acta
1976; 68: 291-301.
Standefer J C; Lenz P H, Wien G H, Toth J M. Triiodothyroninc radicimmunoassay:
incubation time, temperature, and antiscra. Clin Chem 1976; 22: 396-7.
Stepanas A V, Mashiter G, Maisey M N. Serum triiodothyronine: clinical experience
with a new radioimmunoassay kit. Clin Endocrinol 1977; 6:171-83.
Sterling K, Bellabarba B, Newman S I, Brenner M A. Determination of triiodothyroninc
concentration in human serum. J Clin Invest 1969;48:1150-8.
Sterling K, Milch P O. Thermal inactivation of thyroxine-binding globulin for direct
radioimmunoassay of triiodothyronine in serum. J Clin Endocrinol Metab 1974;
38: 866-75.
Surks M J, Schadlow A R, Oppenheimer J H. A new radioimmunoassay for plasma L-
triiodothyronine: measurements in thyroid disease and in patients maintained on
hormonal replacement. J Clin Invest 1972; 51: 3104-13.
Twomey S L, Sweat R. Radioimmunoassay for triiodothyronine: use of polyethylene
glycol to precipitate immune complexes. Clin Biochem 1975; 8: 169-76.
Weeke J, Orskov H. Synthesis of I monolabcled 3,5,3'-triiodothyroninc and thyrox-
ine of maximum specific activity for radioimmunoassay. Scand J Clin Lab Invest
1973; 32: 357-60.
Weeke J, Orskov H. Ultrasensitive radioimmunoassay for direct determination of free
triiodothyronine concentration in serum. Scand J CLin Lab Invest 1975; 35: 237-44.

r /v

'i « (' "v-

 60
//. 3c. ThyroxinefTJ. V..
•rJ,
Murphy and Pattee (1964) introduced a competitive protein binding method
for measurement of T4 in human serum (referred to as T 4 -D,T 4 assayed by
displacement). Essentially, it is based on the competition between unlabeled
T 4 (derived from the unknown serum sample by ethanol extraction) and
labeled T 4 for binding sites on a fixed quantity of added TBG; separation of
the free hormone fraction is effected by adsorption to resin or charcoal. Com-
parison of the results of unknown samples with the standard curve gives the
hormone concentration of the unknown sample. The T 4 radioimmunoassays
I
which were soon developed after the success of the T3-RIA's, were found to m
be superior to the Murphy-Pattee method.
We used the T4-D-method initially (employing the Tetrasorb-125 T 4 diag-
nostic kit purchased from Abbott Laboratories, North Chicago), but changed
to the T4-RIA during our studies. We found almost complete agreement in
the T 4 values measured with both techniques (yx4.RIA = •0-286 +1.064xx 4 .£> •
r=0.9825; n = 145). The T 4 -RIA, essentially the technique of Chopra (1972),
will be described briefly. IS'
3c. 1. Ingredients
a. Buffer-solution. The composition of the buffer is: sodium barbital 0.075
M, merthiolate 10~4M, bovine serum albumin 0.25% in distilled water,
pH=8.6bylMHCl.
b. T^Standard. 3,5,3',5'-tetraiodo-L-thyronine (free acid) was purchased
from Sigma Chemical Company, St Louis, Mo. Standard preparations were I
made by dissolving the agent in 0.01 M NaOH/propylene glycol(l:2), and n
further dilution in buffer. Storage was at -20°C.
c. Tn-Antiserum. Twelve rabbits ("Vlaamse reuzen") were immunized with
bovine thyroglobulin (purchased from Sigma), emulsified in Freund's adju-
vant. All rabbits developed antisera with good avidity to T 4 , although
some antisera had higher T 3 avidity.
d. Tn-Tracer. 1 2 5 I-T 4 is purchased from Abbott Laboratories, North Chica-
go, 111., USA. The specific activity of this preparation is appr. 100 fiCi/ng.
Before use the tracer is purified by Sephadex G-25 chromatography in the
same manner as described for the T3-tracer. The purified tracer is diluted
in buffer to ± 20.000 c.pjn./50/Ltl, and stored at -20°C.
3c.2. Assay procedure.
a. Milieu. Iodothyronine-free plasma prepared by the resin technique (see
section 3a.2a) is employed in the tubes of the standard curve. The tracer
honnone was (mainly non-specifically) bound for 47% in the presence of
salicylate (3mg/tube) as blocking agent, but for 6% when ANS (300 Mg/
tube) was used. ANS was therefore selected as blocking agent.
 b. Tracer concentration. Approximately 120 pg T 4 * per tube (± 20.000
c,p.m./50juI) is added.
c. Antiserum dilution. Antiserum 3045 demonstrated the greatest avidity to
T 4 , and was used exclusively. Excess antiserum binds the tracer for more
than 95% (figure 11.12, upper part). The optimal final dilution of this anti- ' |
serum is 1=2500. %
d. Incubation conditions. The test tubes are incubated for 18-24 hours at |

e. Separation of bound and free liormone fraction. Separation by dextran- j g

' I coated charcoal resulted in a non-specific binding of 20%, and was there-
; I fore abandoned. Polyethylene glycol proved to be very effective as separa-
% ting agent.
f. Assay procedure. Reagents are pipetted in 4 ml polystyrene tubes in the
)\ following order:
\ x n\ buffer up to a final volume of 1 ml
I 50 p\ ANS (300 fig, tube), freshly made
^ 50 nl standard (containing 0-5 ng T 4 /tube) in standard curve tubes only)
L 20 nl iodothyronine-free plasma (in standard cuve tubes only)
f 20 nl unknown plasma
If 50 Ml tracer (containing 20.000 c.p.m., ± 120 pg T 4 ) <
| 50 jul antisenim 3045, FD 1=2500. s
| The tubes are incubated for 18-24 hours at 4°C. Separation is performed • |;
% by PEG, as described under 3a. 2f. Each sample is assayed in triplicate.
,| The percentage bound hormone is corrected for non-specific binding by
1 simultaneously assaying samples in the absence of antiserum.

3c. 3. Results.
':. a. Sensitivity and precision. The detection limit of the assay is 50 pg/tube, or
about 3.2 nmol/1. The intra-assay coefficient of variation is 5%at 62
I nmol/1,6.3% at 83 nmol/1,3.4% at 115 nmol/1, and 4% at 209 nmol/1
;' (each sample assayed in tenfold). '•
lv b. Specificity. Cross-reactivity to related compounds is presented in table :
| 11.17. Dilution curves of unknown plasmas paralleled the standard curve. » {
|; The percentage recovery of exogenously added T 4 is on average 100.9% as ?
determined in 16 experiments ranging from 98 to 104%. It was equal to I
the recovery of 100.2% in thirteen experiments employing ethanol ex- iI
traction of T 4 . Non-specific binding varies from 4 to 7%, and is rather con- : |
stant in the controls of standards and unknowns in one assay. Correction |
for non-specific binding is consequently calculated from the mean of 5 f
controls in one assay. -; 1
c. Quality control. The inter-assay coefficient of variation is 11.8% at 38 i r
nmol/1 (n=28), 6.0% at 86 nmol/1 (n=32), 5.3% at 105 nmol/1 (n=29), | |
3.5% at 148 runol/1 (n=30), and 4.4% at 159 ranol/1 (n=32). } |
Cross-reactivity of thyroid hormone analogs in the T4 radioimmunoassay (AS 3045, FD
1=2500) (see also legend table H.9).

thyroid hormone analog abbreviation % cross-reac- tvity

3,5,3',5' - tetraiodo - L - thyroninc T4 100

I-
3,5,3',5' - tetraiodo - D - thyionine D-T4 100
3,5,3' - triiodo - L - thyronine T3 0.2
3,3' - diiodo - L - thyronine 3,3'-Ta <0.01
3,5,3',5' - tetraiodo - L - thyroacetic acid TETRAC 6.5
3,5,3' - triiodo - L - thyroacetic acid TRIAC 0.3
3,5 - diiodo - L - tyrosine DIT <0.01
3 - monoiodo - L - tyrosine MIT <0.01
thyroglobulin Tg 0.26

3c 4. Discussion.

The T 4 radioimmunoassay is rather easy as compared with T 3 and rT3-RIA's,

due to the relatively high plasma concentration of T 4 . Consequently, there is
no need for great sensitivity and high specific activity tracers, and even con-
siderable cross-reactivity to T 3 does not result in detectable changes in meas-
ured T4-values. The robust character of the assay is reflected in the low intra-
and inter-assay variation, and in the close agreement between the reported
normal values (see table 11.18). Antiserum 3045 is peculiar in demonstrating
a very low cross-reactivity to T 3 , but this is of no practical advantage. Most
assays reported in the literature employ ANS to displace T 4 from TBG. It
should however be realized that in plasmas with high TBG concentrations
ANS may not completely inhibit the T4-TBG binding, resulting in high values
when polyethylene glycol and double-antibody methods are used for separa-
tion of the bound hormone fraction (Evans et al 1977). Recently, the effect-
iveness of ANS as a competitor for thyroid hormone binding sites has been
explained by structural similarities between the molecular conformation of
ANS and thyroid hormones (Cody and Hazel 1977).
The T4-RIA has been found to be superior to the T4-D technique in many
respects. It obviates the extraction step, and due to its greater sensitivity
much less plasma is needed than in the T4-D method. The linear range of the
standard curve is from 1S-S18 nmol/1 in the T 4 -RIA, and from 39-259 nmol/1
in the T4-D (Chopra 1972), permitting more samples to be assayed undiluted
by T4-RIA. The T4-RIA is also more specific, since halofenate (Karen et al
 63
1976), unesterified fatty acids (Rootwelt 1975; Shaw et al 1976) and orphen-
J1.-''
adrine (Wiersinga and Touber 1977) all interfere in vitro in T4-D assays but
not in T4-RIA's. These agents or its metabolites are soluble in ethanol, and I
the ethanol extracts will contain appreciable quantities of these ethanol-
soluble compounds, which compete with labeled thyroxine for the binding
sites on the fixed quantity of TBG added in the T4-D assay, thus leading
to spuriously elevated 'thyroxine" values. Applying the T 4 -D method in
serum extracts obtained by Sephadex chromatography results in normal
values, just as in the T4-RIA.
There is a very good agreement in T 4 values as measured by T4-RIA and
by the T4-D assay for the hyperthyroid and euthyroid range, but many
authors report occasionally higher values by RIA than by D-assay for samples
in the hyperthyroid range. The discrepancy disappears when the RIA is ap*
plied to serum extracts (Chopra 1972). According to Chopra (1972) these
inconsistences are due to the presence in serum of T 4 covalently linked to
serum proteins; the nature of this nonextractable T 4 is unlikely to be thyro- I,
globulin. Herrmann (1973) and Kubasik (1973) point to the. decrease in
recovery found at high T 4 concentrations in the T4-D method but not in the
T4-RIA. Seth (1973) found the same T4-values in unextracted serum and in
Sephadex-extracted serum, which also negates the presence of significant
quantities of T 4 covalently linked to protein. Different separation techniques
in the T4-RIA were applied by Premachandra (1976); although mean T 4 -
values did not differ with the various separation techniques used, individual
I
samples demonstrated considerable variation. The occasionally found dis- u
crepancy between T4-values of hyperthyroid patients measured by RIA or
the D-method, seems to be a methodological problem, probably resulting
from incomplete extraction of T 4 in the T4-D assay and from incomplete
displacement of T 4 from TBG by ANS in the T4-RIA.

3c. 5. References.

Beckers C, Cornette C, Thalasso M. Evaluation of serum thyroxine by radioimmuno-

assay. J Nud Med 1973; 14: 317-20.
Chopra 1 J, Solomon D H, Ho R S. A radioimmunoassay of thyroxine. J Clin Endo-
crinol Metab 1971; 33: 865-8.
Chopra I J. A radioimmunoassay for measurement of thyroxine in unextracted serum.
J Clin Endocrinol Metab 1972; 34: 938-47.
Cody V, Hazel J. Molecular confirmation of ammonium 8-aniIino-l-naphthalene-sulfo-
nate hemihydrate. A fluorescent probe for thyroxine binding to thyroxine binding
globulin. J Med Chem 1977; 20:12-6.
Dunn R T, Foster L B. Radioimmunoassay of thyroxine in unextracted serum, by a
single-antibody technique. Clin Chem 1973; 19:1063-6.
Evans S E, Burr W A, Hogan T C. A reassessment of 8-anilino-l-naphthalene sulphonic
acid as thyroxine binding inhibitor in the radioimmunoassay of thyroxine. Ann Clin
Biochem 1977; 14: 330-4.
 Table 11-18
Main features of T 4 radioimmunoassays, as reported in the literature

REFERENCE TRACER ANTISERUM T4-TBP INCUBATION SENSI- % CROSS-REAC-

NEUTRAL- TIVITY TIVITY'
IZATION
specact PgT 4 immuni- FD incubation separation nmol/I ' !• T , '„ ''' _', '•'',;
MC/pg added zation B/F ' • > • • : ' ; • • • ' • ' . •

Chopra 1972 50-75 200 hTg 1=300 ANS 150pg 1 hr room DA 15 8.4
temp. 5 min.
4°C
Mitsuma 1972 50 T4-BSA 1=1000 ANS 175ug 90' 37°C charcoal 0
10' 40°C
Larsen 1973 bTg 1=2000 salicylate 18 mg/ml overnight 4°C, charcoal 10.9
or:90'37 Q C +
20' 4°C
Beckers 1973 50-80 200 bTg 1=500 ANS 300pg pre-incub: DA 13
i
1 hr room temp.
l'hr room temp.
Dunn 1974 30-50 700 T4-BSA heat 1 hr room temp. charcoal 3 . 4 •:. -
denaturation =
boiling 1 hr.
Meinhold 1974 80-100 50 T4-HSA 1=1800 I ANS300pg 20hrs. 4°C DA 5
1
Werner 1974 100 1=3000 [ ethanol extraction 30' 40° C PEG ; <10.0
Herrmann 1974 100-170 10-30 T4-BSA 1=2000 ANS600pg 90' 37° C charcoal 3; • 2.1
10' 4°C
Ratcliffe 1974 20-50 500 T4-HSA 1=5000 ANS 400jug 2 hr 37°C DA 5''.' , ' " ' 3 . 5 ' • ••,•'•
400-800 or overnight 4°C ! (1.1-5.2)
Seth 1975 T4-BSA 1=4500 ANS 24 hrs. 4°C antibody 10 ! 1 . 3 :
•;•••
1 1 '< ' '
1=6000 wash solu- ' ' ; '

tion 1
• ' ' ' . "

Visser 1975 500 bTg 1=4000 ANS 350/ig 16 hrs. 4°C DA 6 '' : '' 0 . 6 ' ' ''• "i
Marsden 1975' 25-50 25 T4-BSA 1=500 ANS 200jug IV, hr 37° C charcoal ! 1.6 ,, ! . ' ' ! • ! 8 , ' -
Prema- 1976 335 bTg 1=157 TCA-NaOH 1 hr 37°C resin 8 4
chandra 1 15' room temp. '. '
Farid 1977 ANS 300Mg 2 hrs room temp.PEG <13 0.6
Wiersinga 1979 100 120 bTg 1=2500 ANS 300Mg 18-24 hrs 4° C PEG 3 V) ! 0.2 :: : • , ;

.>.•• ..- .y
 \ i • I; I , . . - . . *

REFERENCE NORMAL VALUES HYPERTHYROIDISM HYPOTHYROIDISM

x± SD range n x + SD range n x±SD range n

Chopra 1972 108 ± 31 5 8 - 171 40 323t 113 1 5 3 - 596 40 45 ±12 30-62 7

Mitsuma 1972 96 ±25 5 1 - 127 15 210± 47 1 5 6 - 285 11 17 ± 10 5-26 14
Larsen 1973 108 ± 21 59 264± 84 40 26 ±17 32
Beckers 1973 124 ± 26 79 327 ± 104 30 34 ± 2 0 21
Dunn 1974 5 8 - 153 54
Meinhold 1974 104 ± 21 7 1 - 152 25 1 7 9 - 443 15 5-75 15
Werner 1974
Herrmann 1974 235 * 48 62
Ratcliffe 1974 100 ± 21 5 6 - 145 61 249± 48 1 7 1 - 332 23 18 ± 9 < 5-34 17
Seth 1975
Visser 1975 110 ± 25 36 242± 54 20 17 ± 1 8 13
Marsden 19751 81 ± 1 4 30 183 ± 55 18 8± 9 14 overt
52 ±25 4 borderline
Prema-
chandra 1976 104 ± 21 7 1 - 161 no 242± 62 1 6 5 - 368 58 32 t 17 0-71 43
Farid 1977 114 ± 19 96 235± 60 9 49 ±25 11
Wiersinga 1979 102 ± 18 6 2 - 151 63 197± 50 123 - 338 55 26 ± 2 0 < 5-68 33
123± 13 / 1 0 7 - 145 11'
1
no iodothyronine-frce serum in standard curve
'Tj -toxicosis
3
on a molar base
 66
Farid N R, Kennedy C. Assessment of a method for measuring serum thyroxine by radio-
immunoassay, with use of polyethylene glycol precipitation. Clin Cheui 1977; 23:
1333-S.
Herrmann J, Rusche H J, Kriiskemper H L. Rapid radioimmunoassay for the evaluation
of thyroxine in unextracted serum. Clin Chim Acta 1974; 54:69-79.
Karch F E, Morgan J P, Kubasik N P, Sine H E. Effect of halofenate on serum thyroid
hormone determinations in vitro. J Clin Endocrinol Metab 1976; 43: 26-31.
Kubasik N P, Sine H E, Murray M H. Serum thyroxine: results compared for a commer-
cial radioimmunoassay and a commercial competitive protein binding analysisrCJin
Chem 1973; 19:1307-08.
Larsen P R, Dockalova J, Sipula D, Wu F M. Immunoassay of thyroxine in unextracted
human serum. J Clin Endocrinol Metab 1973; 37:177-182.
Marsden P, Facer P, Acosta M, Howorth P J N. A comparative study of serum total
thyroxine estimation on unextracted serum by radioimmunoassay and by com-
petitive protein binding. J Clin Path 1975; 28:608-12.
Meinhold H, Wenzel K W. Radioimmunoassay of thyroxine in unextiacted serum. Horm
Metab Res 1974;6:169-170.
Mitsuma T, Colucci J, Shenkman L, Hollander C S. Rapid simultaneous radioimmuno-
assay for triiodothyronine and thyroxine in unextracted serum. Biochem Biophys
Res Commun 1972; 46: 2107-13.
Murphy B E P, Pattee C J. Determination of thyroxine utilizing the property of protein-
binding. J Clin Endocrinol Metab 1964; 24:187-96.
Ptemachandra B N, Ibrahim II. A simple and rapid thyroxine radioimmunoassay (T 4 -
RIA) in unextracted human serum; a comparison of T4-RIA and T 4 displacement
assay, (T 4 -D), in normal and pathologic sera. Clin Chim Acta 1976; 70: 43-60.
Ratcliffe W A, Ratcliffe J G, McBride A D, Harland W A, Randall T W. The radioimmu-
noassay of thyroxine in unextracted human serum. Clin Endocrinol 1974; 3: 481-8.
Rootwelt K. The influence of fatty acids on serum thyroxine determination* by compet-
itive protein-binding radioassay. Scand J Clin Lab Invest 1975; 35: 649-53.
Seth J, Rutherford F J, McKenzie I. Solid-phase radioimmunoassay of thyroxine in un-
extracted serum. Clin Chem 1975; 21:1406-13.
Shaw W, Hubert I L, Spiertoo F W. Interference of fatty acids in the competitive pro-
tein-binding assay for serum thyroxine. Clin Chem 1976; 22: 673-8.
Visser T J, van den Hout-Goemaat N L, Docter R, Hennemann G. Radioimmunoassay
of thyroxine in unextracted serum. Neth J Med 1975; 18:111-115.
Werner S C, Acebedo G, Radichevich I. Rapid radioimmunoassay for both T 4 and T s in
the same sample of human serum. J Clin Endocrinol Metab 1974; 38: 493-5.
Wiersinga W M, Fabius A J M,Touber J L. Orphenadrine (Disipal), serum thyroxine and
thyroid function. Acta Endocrinol 1977- 86: 522-32.

t
 6 7
?;••
Sc H.4. Traodothyronme uptake ratio (T3 U)

Y-r- In the blood thyroid hormones are bound t o plasma proteins, predominantly
••- • to thyroxine-binding globulin (TBG), but also to albumin and a thyroxine- ;
^ binding prealbumin (TBPA). T 3 is bound to TBG and albumin, but T 4 and 5
v
I rT3 also to TBPA (Snyder et al 1976; Chopra 1978). TBG has the highest j
affinity for T 4 and the lowest for T 3 , and serves as a high-affinity, low i
capacity binding protein, whereas albumin and TBPA have a low-affinity
high-capacity binding for iodothyronines. Therefore, under normal circum- 1 <
stances TBG is the most important thyroid hormone binding protein in the
blood. The proportion of unbound thyroid hormone in the blood is 0.03%
for T 4 , and 0.3% for T 3 and rT 3 . It is generally accepted that only unbound
thyroid hormones can enter the target tissues, and it is the free thyroid hor- ' s ;
;
mone concentration in the blood which is of major importance in deter-
mining the metabolic state of the peripheral tissues. Due to the very low con- ,
centration of free thyroid hormone it is not easy to measure it directly. An ;
indirect estimation of the free thyroid hormone concentration can however .
be easily obtained.
The dynamic equilibrium between free and bound thyroid hormone in t
plasma can be expressed as (T 4 is chosen as the prototype of iodothyronines,
and TBG as the most important binding protein) (Woeber 1978):
;
T 4 +TBG ^ T 4 . TBG
According to the law of mass action it follows:
[T 4 . TBG]
[T4].[TBG]~K
in which [T 4 . TBG] represents the concentration of T 4 bound to TBG,
[T 4 ] the free T 4 concentration, [TBG] the concentration of unoccupied •;
binding sites on TBG, and K the equilibrium constant. This expression can
be rearranged as:
£ K. [T4] = [T 4 . TBG] . — — ':
I [TBG]
I The radioimmunoassay of T 4 described under section 3c of this chapter,
I measures the total T 4 concentration in plasma, and this can be considered •
I equivalent to [T4.TBG] since the unbound fraction of T 4 is very small. ';
I Measurement of——can easily be done with the triiodothyronine uptake :
I [TBG] \
i ratio (T 3 U), a procedure originally introduced by Sterling and Tabachnick [\
t (1961). Radiolabeled T 3 (preferred over labeled T 4 due to its lower affinity ;|
I for TBG) is incubated with a plasma sample and an insoluble, particulate %
I material (usually an anion exchange resin). The uptake by the resin of any 3
I labeled T 3 not bound to the plasma proteins, is determined (hence the term \

•••••*—r.y.
 "• u .

68
'resin T 3 uptake' or T 3 U). Since the labeled T 3 is bound to the plasma -a"

proteins and to the resin in the same proportion as the endogenous hormone,
the resin T 3 uptake varies inversely with the binding affinity and concentra- vv.
tion of unoccupied binding sites on the plasma proteins. It thus represents the
proportion of free thyroid hormone in plasma.
From the product of total T 4 concentration and the T 3 U in a plasma
I
sample a free thyroxine index (FT 4 index) can be calculated (Clark and Horn ¥
1965). The FT 4 index is in good correlation with the free T 4 concentration
(measured by equilibrium dialysis) in normal euthyroid adults, in hyper-
thyroid patients and in hypothyroid patients, but not in acutely or severely
ill euthyroid patients ("sick euthyroid patients") (Anderson 1969). In prin-
ciple, by the same procedure FT 3 and FrT 3 indices can be obtained, and a
good correlation between the FT 3 index and the free T 3 concentration has
been found in normal euthyroid subjects and in hypothyroid or hyperthyroid
patients (Olsen and Andersen 1979).
As described above, the free thyroxine concentration is positively related
to the total T 4 concentration, and negatively to the binding affinity and il
concentration of unoccupied binding sites of TBG for T 4 . It thus is subject
to changes in TBG concentration and to changes in T 4 supply (Woeber 1978).
An increased T 4 supply (e.g. in hyperthyroidism) results in a greater total
T4 concentration and a greater proportion of unbound T 4 , i.e. in a greater
free thyroxine concentration; the reverse is true for a decreased T 4 supply
as in hypothyroidism. An increase in TBG concentration (e.g. induced by
estrogens) results in a greater total T 4 concentration and in a smaller propor-
tion of unbound T 4 , i.e. the free thyroxine concentration does not change.
In general , concordant changes in T 4 and T 3 U tests indicate a change in
hormone supply, whereas discordant changes indicate changes in hormone
binding to the plasma proteins (see table 11.19).
The T 3 U method used in our laboratory is the Triosorb M-125 kit pur-
chased from Abbott Laboratories, North Chicago. The inter-assay coefficient
of variation of T 3 U is 4.4% at a ratio of 0.90 (n=10), 3.0% at a ratio of 1.01
(n=9), and 3.6% at a ratio of 1.11 (n=8). The results are expressed as the
triiodothyronine uptake ratio, that is the T 3 U of the unknown plasma
sample divided by the T 3 U of a pool of normal standard reference serum
included in the same assay run. This form is strongly preferred over expres-
sion of the results as percentage uptake of the labels on the resin, and this
form should be used exclusively in calculating a free thyroxine index (The
Committee on Nomenclature of the American Thyroid Association 1976).
By doing so the normal range of T 3 U is centered on 1.0, and the calculated
FT 4 index is of the same order as the total T 4 concentration. Whereas many
physicians have already difficulties in distinguishing total T 3 concentration
measured by RIA from the T 3 U (both are frequently referred to as T 3 test),
confusion has been complete by the use of the inverted fraction of T 3 U in
 69
•*•'.

Factors that influence free thyroxine concentration, according to Woeber (1978). (t = in-
I?'' creased; i = decreased; N = normal).

total T4 concentration % free T4 concentration of free T4

or or or
factor T4-RIA, T4-D T3U FT 4 index
i increased T4 supply
decreased T4 supply
increased T4 binding N
decreased T4 binding N

some methods, resulting in low ratios in hyperthyroidism and high ratios

in hypothyroidism. This should be abandoned, and the notation of theT 3 U
should always be in such a manner that hyperthyroidism is associated with
increased ratios and hypothyroidism with decreased ratios.

References

Anderson B G. Free tliyroxine in serum in relation to thyroid function. JAMA 1968;

203:135-40.
Chopra I J. Nature, source and biologic significance of thyroid hormones in blood. In:
Werner S C, Ingbar S H, eds. The thyroid, 4th edition. Hagerstown: Harper and Row,
1978: 100-14.
Clark F, Horn D B. Assessment of thyroid function by the combined use of the serum
protein-bound iodine and resin uptake of I-triiodothyronine. J Clin Endocrinol
Metab 1965; 25: 39-45.
The Committee on Nomenclature of the American Thyroid Association. Revised nomen-
clature for tests of thyroid hormones in scrum. J Clin Endodrinol Metab 1976; 42:
595-8.
Olsen K J, Anderson C J. Determination of free thyroid hormone fractions using a
simple T3-uptake test. Scand J Clin Lab Invest 1979; 39:119-23.
Snydcr S M, Cavalieri R R, Goldfine ID, Ingbar S H, Jorgensen E C. Binding of thyroid
hormones and their analogs to thyroxine-binding globulin in human serum. J Biol
Chem 1976; 251: 6489-94.
Sterling K, Tabachnik M. Resin uptake of I131-triiodothyronine as a test of thyroid
function. J Clin Endocrinol Metab 1961; 21: 456-64.
Woeber K A. Tests of thyroid hormone transport. In: Werner S C, Ingbar S H, eds. The
thyroid, 4th edition. Hagerstown: Harper and Row, 1978: 33844.
^

if
 70
II. 5. Sample handling and storage.

a. Plasma versus serum samples. Assays were done in plasma and in serum
samples obtained simultaneously from the same subject (table 11.20). The
results are the same in all assays whether plasma or serum is used; this is in ac-
cordance with other studies (Kubasik 1978). For reasons of convenience it fc. •
was therefore decided to routinely collect blood in heparinized tubes (Searle
LH/10, lithium heparin) and to use plasma exclusively.
b. Time of sampling. Blood was collected in the morning between 8.30 and
10.30 pjn. in semirecumbent position, unless stated otherwise. This
procedure avoids interference of diurnal variation in comparing hormone
levels (De Costre et al 1970; Weeke 1973).
c. Technique of blood collection. Thyroid hormones in the blood are bound
to proteins for more than 99%. Thus, venous stasis is likely to induce
increased values. The effect of various techniques of blood sampling
was evaluated in 11 patients. Samples were withdrawn (a) without venous
stasis by an indwelling venous catheter (b) with venous stasis by ordinary
venapuncture in the contralateral arm (c) after 5 minutes of venous stasis
through the indwelling venous catheter. The three samples in each patient
were obtained within 7 minutes. As presented in table II.21, venous stasis is
associated with an increase in the values of iodothyronine plasma concen-
trations; FT 4 index does not change due to the concomitant decrease in
T 3 U. These changes are related to the increase in protein concentration. The
same observations have been made by several authors (Lalla and Grasbeck
1975; Judd et al 1975; Jacobsen et al 1977). Our experience is that in practice
venous stasis can pose problems, since not infrequently elevated plasma
T 3 concentrations in patients referred under the diagnosis of T3-toxicosis,
were found to be exclusively caused by longstanding venous stasis due to a
difficult venapuncture.
d. Storage of plasma samples. Plasma samples are stored at -20°C until assay.
The samples are frozen within 4-6 hours after the blood collection. We

Table 11-20
Comparison of plasma and serum in the assays of TSH, rT 3 , T 3 , T4 and T3 U (values as
meantSD).

assay n plasma serum

TSH mU/1 25 1.65 ± 0.9 1.59 ± 0.8

rT, nmol/l 14 0.23 ± 0.09 0.24 ± 0.09 i
T3 nmol/l 24 1.61 ± 0.35 1.62 ± 0.34
T« nmol/l 25 105 ± 18 105 ± 16
T3U 20 0.973 ± 0.10 0.977 ± 0.10
Table 11-21
Effect of venous stasis on the plasma concentration of TSH, rT3, T, and T 4 , and on T3 U and FT4 index in 11 patients (values as mean ± SD, or
as geometric mean with x-SD and x+SD between brackets; statistical significance of (b) vs (a) and of (c) vs (a) by the paired t test).

assay without venous stasis, with venous stasis after 5 min. venous stasis.
by indwelling catheter by venapuncture by indwelling catheter
(a) (b) p(b vs.a) (c) p(c vs.a)
Hb mmol/1 8.3 ± 1.1 8.6 ± 1.1 <0.01 8.7 ± 1.1 <0.02
Htl/1 0.407 ± 0.06 0.417 ± 0.05 <0.02 0.422 ± 0.06 <0.05
total protein g/1 71.8 ± 5.4 73.1 ± 3.7 N.S. 76.6 ± 4.7 <0.01
albumin g/1 42.4 ± 3.3 42.5 ± 3.1 N.S. 44.2 ± 4.0 <0.01
TSH mU/1 1.2 ( 0.3 - 7.8 ) 1.1 ( 0.3. - 7.4) N.S. 1.1 ( 0.3 - 7.2 ) N.S.
rT3 nmol/1 0.28( 0.14- 0.57) 0.30( 0.14- 0.63) N.S. 0.32( 0.16- 0.63) <0.05
T3 nmol/1 2.12( 1.11- 4.05) 2.13( 1.13- 4.01) N.S. 2.24( 1.18- 4.26) <0.05
T4 nmol/1 80 (46 -137 ) 84 (49 -145 ) <0.05 87 (49 -154 ) <0.05
T3U 1.06( 0.81- 1.38) 1.04( 0.81- 1.33) <0.05 1.01( 0.79- 1.29) <0.01
FT4 index 85 (41 -177 ) 87 (42 -180 ) N.S. 88 (41 -185 ) N.S.
 Seth J, Toft A u, Irvine w J. sunpie sguu-piiasc iauiui

72
have not encountered appreciable changes in hormone concentrations of
TSH, T4 andT 3 stored at -20°C for 3 years.
I References.
jy: De Costre Ph, Buhler U, De Groot L J, Refetoff I. Diurnal rhythm in total serum thyrox-
ine levels. Metabolism 1971; 20: 782-91.
Jacobsen B B, Petersen B, Andersen H. Changes in serum level of thyroxine and thy-
roxine-binding proteins (TBG, TBPA, albumin) induced by venous stasis. Acta Endo-

i crinoll977; 85: 39-43.

Judd, SJ, Carter JN, Corcoran JM, Lazarus L. Circulating thyroid hormone concen-
trations and posture and venous compression. Brit Med J 1975; 4: 735-6.
5-1
Kubasik NP, Sine H E. Results for serum and plasma compared in 15 selected radio-
assays. Clin Chem 1978; 24:137-9.
Lalla M, GrSsbeck R. Effect of tourniquet application, muscle work and vacuum tube
blood collection on serum concentrations of Na, K, creatinine, total protein, ASAT,
ALAT, cholesterol, PBI, T 3 U and triiodothyronine. Scand J Clin Lab Invest 1975;
Suppl. 143: 101.
Weeke J. Circadian variation of the serum thyrotropin level in normal subjects. Scand
J Clin Lab Invest 1973; 31: 337-42.

h
CHAPTER HI.

STUDIES IN EUTHYROID VOLUNTEERS

///. 1. Introduction.

Normal values for the assays of TSH, T 4 , T 3 , rT3 and T3 U were determined
in euthyroid volunteers and the effect of physiological factors as age, sex and
weight on these parameters was evaluated. Stimulation with thyrotropin-
releasing-hormone (TRH) was performed in all volunteers, and the normal
response was determined. The TRH-test was included in our studies because
of its great value in the diagnosis of thyroid function disorders (a positive
TSH response to TRH virtually excludes the diagnosis hyperthyroidism), and
because of its sensitivity to very small changes in thyroid hormone concen-
tration in the plasma(Snyder and Utiger 1972; Vagenakis et al 1974).

///. 2. Subjects and methods.

In all subjects an indwelling venous catheter was inserted in an antecubital

vein, and blood sampling was delayed until IS minutes after the insertion of
the catheter. T4 was determined initially by the displacement method of
Murphy-Pattee, and subsequently by radioimmunoassay. The TRH prepara-
tion used was kindly provided by Dr. W J. van Rijn (Relefact TRH, Hoechst
Holland); in all cases 200 y% TRH was rapidly injected intravenously.
In six fasting volunteers (3 women, 3 men, mean age 32 years, range 2248
year) control tests were performed: these subjects were injected intravenously
at two occassiCiis with 200 jug TRH resp. physiological saline; the interval
between the two tests was at'least one week.
Sixty-three subjects were tested in order to determine the range of normal
basal values and the normal response after TRH stimulation. These subjects
were considered to be euthyroid on clinical grounds. Subjects were excluded
for one or more of the following reasons: history of thyroid disease, goitre on
physical examination, grossly abnormal body weight, medication known to
influence thyroid function tests, acute or chronic disease. No attention was
given to the time of testing with respect to the menstrual cycle. Healthy
people of 60 years and older all resided in an home for the elderly in Amster-
dam. The data for sex, age, weight and height in these normal adults are pre-
sented in table III. 1. From the 63 subjects 25 were tested after an overnight
fast, and 38 were tested after breakfast.
Table III-l
Age, weight and height (given as mean ± SO, with range between brackets) of the normal adult population, distributed to sex, age and season
in which blood sampling was performed.
number age (years) weight (kg) height (cm)
9 d total
sex 9 32 0 32 42.7 ± 15.0(22-88) 61.9 ± 6.2(44.5-73.0) 166 ± 6(154.5-177.5)
6 0 31 31 49.6 ± 17.4(24-86) 75.01 7.5(56.5-89.0) 175 ±6(161 -189 )
all subjects 32 31 63 46.1 ± 16.4(22-88) 68.4 ± 9.5(44.5-89.0) 171*8(154.5-189 )

age 20 - 29yr 7 3 10 25.8 ± 2.4(22-29) • 66.0 ± 12.8(44.5-88.5)

30 - 39yr 7 7 14 34.7 ± 2.8(30-39) 66.6 ± 8.7(47.5-77.0)
40 - 49yr 9 9 18 45.1 ± 2.6(40-48) 69.5 ± 9.5(54.0-89.0)
50 - 59yr 6 5 11 53.4 ± 2.8(50-57) 69.3 ± 9.8(58.7-86.0)
> 60yr 3 7 10 76.0 ± 10.1(60-88) 70.2 ± 7.0(62.0-82.0)

season winter 5 13 18 44.7 ± 11.3(23-64) 72.5 ± 9.4(56.5-86.0)

spring 3 6 9 42.0 ± 11.9(27-63) 68.3 ±. 6.4(60.0-77.0)
summer 22 8 30 47.4 ± 21.4(22-88) 66.2 ± 9.9(44.5-89.0)
fall 2 4 6 44.7 ± 2.6(41-48) 66.6 ± 9.1(54.0-78.0)
 75
Nineteen cord blood samples from healthy newborns were also analysed.
The basal value of the thyroid function tests in each individual was calculated
as the mean of the two samples taken before the injection of TRH. In the
statistical analyses undetectable hormone concentrations were taken as SO
percent of the detection limit; the level of significance was taken as a = 0.05.
Differences between subgroups of the normal population are evaluated by
Willcoxon's two sample test. In the determination of normal limits preference
is given to warning limits over descriptive limits, since the risk that unusual
values remain undetected should be kept small in the diagnostic process. The
inner limits for the percentiles P2.5 and P97.5 with a reliability y = 90% are
therefore determined according to a distributionfree method (Riimke and
Bezemer 1972), and these warning limits constitute the nonnal limits.
The hormone response to TRH stimulation was calculated as the differen-
ce between the observed peak value after TRH and the mean of the two basal
values. This approach is more practical than calculating the surface area.

227.3. Results.

a. Basil values
1. General. There was a non-parametric distribution of all parameters un-
der test (figure III.l); after logarithmic transformation the distribution
of the plasma iodothyronine concentrations appeared to be nonnal.

n
20
16 TSH rT,
12

0
03 1-0 2.0 00 4.0 60 0 0.1 0.2 03 0.4 0.5 1.0 1.4 18 22 2.6 3.0
nmol/l ,_. nmol/l
20

16 T,U FT 4 index

12

0
60 80 WO 120 140 160 0.75 O85 095 1.05 1.15 125 60 80 100 120 140 160
nmol/l

Figure III, 1. Distribution of TSH, rT 8 , T , , T 4 , T,U and FT4 index values in 63 normal
euthyr jid adults.
 Effect of sex on the values of thyroid function tests in £3 normal adults (32 females, 31 males).

geometric mean x±SD median range inner limits

(x-SD, x + SD) P2.5 and P97.5

TSHmU/1 9 0.7 (<0.3 - 2.3 ) 1.2 ± 1.0 1.1 <0.3 -4.3

d 0.7 « 0 . 3 - 2.3 ) 1.2 ± 1.2 1.0 <0.3 -4.5
total 0.7 « 0 . 3 - 2.3 ) 1.2 ± 1.1 1.1 <0.3 -4.5 <0.3 -3.1
rT,nmoI/l 9 0.22( 0.16 - 0.30 ) 0.23 ±0.08 0.22 0.11 - 0.44
d 0.23( 0.17 -0.31 ) 0.24 ± 0.07 0.23 0.14 - 0.40
total 0.22( 0.16 - 0.31 ) 0.24 ± 0.07 0.22 0.11 - 0.44 0.14 -0.38
T,nmol/1 9 1.74( 1.41 - 2.16 ) 1.78 ±0.38 1.72 1.00 - 2.78
d 1.88( 1.58 - 2.25 ) 1.91 ±0.35 1.81 1.30 - 2.85
total 1.81( 1.48 - 2.21 ) 1.85 ±0.37 1.75 1.00 - 2.85 1.30 -2.45
T4 nmol/1 9 98( 82 - 118) 100± 18 99 62 - 141
d 103( 88 - 121) 104 ± 17 105 70- 151
total 101( 85 - 119) 102 ± 18 101 62 - 151 75 - 135
TSU 9 0.93( 0.86 - 1.02 ) 0.94 ±0.08 0.93 0.75 - 1.09
d 0.95( 0.87 - 1.04 ) 0.95 ±0.09 0.95 0.81 - 1.19
total 0.94( 0.86 - 1.02 ) 0.94 ±0.09 0.93 0.75 - 1.19 0.81 -1.07
FT4 index 9 92( 77 - 109) 93 ± 16 92 66 - 135
d 98( 85 - 113) 99 ± 14 96 70 - 128
total 95( 80 - 111) 96 ± 15 95 66 - 135 70 - 122
rT 3 /TjXl0- 3 9 126{ 86 - 184) 135 ± 56 119 60 - 314
d 122( 85 - 174) 129± 47 128 60 - 277
total 124( 86 - 179) 133 ± 51 121 60 - 314 69 -- 209
s
rT,/T 4 xl0- 9 2.24( 1.60 - 3.13 ) 2.40 ±0.80 2.13 1.02-4.29
d 2.22( 1.63 - 3.04 ) 2.30 ±0.70 2.30 1.23 - 3.53
total 2.23( 1.62 - 3.08 ) 2.35 ±0.76 2.20 1.02 - 4.29 1.38 -3.53
3
Tj/T 4 xl0" 9 17.8 (14.5 -21.9 ) 18.2 ± 4.0 18.0 11.7 - 3 2 . 2
d 18.3 (15.3 -21.9 ) 18.6 ± 3.2 18.9 11.6 -24.7
all 18.0 (14.9 - 21.9 ) 18.4 ± 3.6 18.2 11.6 - 3 2 . 2 12.8 -23.4
 Table III-3
Effect of age on the values of thyroid function tests in 63 normal adults (20-29 year,
n=10; 30-39 year, n=14; 40-49 year, n=18; 50-59 year, n=ll; >60 year, n=10).

age (years) geometric mean x± SD median range fel"

( x - S D . x + SD)
TSH mU/1 20- 29 0.6 « 0 . 3 - 1.8 ) 0.9 ±0.6 1.15 <0.3 - 1.7
30- 39 0.4 « 0 . 3 - 1.3 ) 0.8 ± 1.0 0.4 <0.3 - 3.5
40- 49 1.3 «0.5 - 3.8 ) 1.9 ±1.3 2.0 <0.3 - 4.5
£•-
50- 59 0.7 « 0 . 3 - 2.5 ) 1.2 ± 1.1 0.9 <0.3 - 2.8
> - 60 0.6 « 0 . 3 - 1.8 ) 0.9 ±0.7 1.0 <0.3 - 1.8
tT3nmol/l 20- 29 0.21(0.14 - 0.32) 0.23 ± 0.10 0.22 0.11 - 0.44
30- 39 0.23(0.17 - 0.30) 0.23 ± 0.07 0.22 0.15 - 0.40
40- 49 0.22(0.16 - 0.30) 0.23 ± 0.07 0.23 0.14 - 0.38
50- 59 0.22(0.16 - 0.31) 0.23 ± 0.08 0.21 0.14 - 0.39
> 60 0.25(0.20 - 0.31) 0.25 ± 0.06 0.24 0.17 - 0.36
T 3 nmol/1 20- 29 1.96(1.62 - 2.37) 1.99 ± 0.40 1.84 1.60 - 2.45
30- 39 1.78(1.50 - 2.12) 1.81 ± 0.31 1.76 1.30 - 2.35
40- 49 1.77(1.40 - 2.24) 1.82 ± 0.43 1.71 1.00 - 2.85
50- 59 1.98(1.75 - 2.24) 1.99 ± 0.24 2.08 1.68 - 2.34
> 60 1.61(1.31 - 1.98) 1.64 ± 0.32 1.67 1.05 - 2.13
T4nmol/1 2 0 - 29 100( 83- 122) 102 ± 20 100.5 75- 141
3 0 - 39 104( 91- 120) 105 ± 14 111 77- 121
4 0 - 49 96( 79- 118) 98 ± 20 96 62- 151
5 0 - 59 103( 90- 117) 103 ± 14 100 85- 135
> 60 101( 83- 123) 103 ± 19 103 70- 137
T3U 2 0 - 29 0.92(0.84-1.01) 0.92 ± 0.08 0.925 0.81 - 1.09
3 0 - 39 0.96(0.91 - 1.02) 0.97 ± 0.07 0.96 0.84 - 1.07
4 0 - 49 0.96(0.86 - 1.07) 0.96 ±0.10 0.96 0.75 - 1.19
5 0 - 59 0.93(0.85 - 1.02) 0.94 ± 0.08 0.92 0.81 - 1.08
> 60 0.90(0.85 - 0.95) 0.90 ±0.06 0.90 0.80 - 1.00
FT4 index 20-29 93( 75- 114) 94 ± 20 91 69- 135
3 0 - 39 101( 88- 116) 101 ± 13 104.5 77- 122
4 0 - 49 93( 77- 111) 94 ± 17 92.5 66- 127
5 0 - 59 96( 85- 108) 96 ± 13 92 83- 128
> 60 91( 78- 105) 91 ± 13 93 70- 109
r T 3 / T 3 x l 0 - 3 2 0 - 29 109( 7 9 - 152) 115 ± 38 107 69- 175
3 0 - 39 126( 8 8 - 180) 134 ± 46 125 64- 209
4 0 - 49 124( 8 3 - 184) 133± 51 127 60- 250
5 0 - 59 111( 81 - 152) 116± 40 106 76- 189
> 60 154( 105 - 225 ) 130 ± 70 140 99- 314
rT 3 /T 4 xl0" 3 2 0 - 29 2.14(1.45 - 3.14) 2.27 ± 0.79 2.20 1.02 - 3.53
3 0 - 39 2.16(1.58 - 2.97) 2.27 ± 0.78 2.27 1.30 - 4.29
4 0 - 49 2.28(1.61 - 3.24) 2.41 ± 0.82 2.30 1.23 - 4.07
5 0 - 59 2.14(1.61 - 2.83) 2.22 ± 0.69 1.92 1.59 - 3.48
> 60 2.46(1.86 - 3.26) 2.55 ± 0.74 2.44 1.72 - 4.02
T 3 /T 4 xl0" 3 2 0 - 29 19.5(15.9 - 24.0) 19.9 ± 4.7 19.6 14.8 - 32.2
3 0 - 39 17.1(14.1 - 20.1) 17.4 ± 3.4 16.9 14.1 - 24.7
4 0 - 49 18.4(15.8 - 21.5) 18.6 ± 2.9 18.3 13.4 - 25.4
" 50 - 5 9 19.3(16.8 - 22.1) 19.4 ±2.6 18.4 15.5 - 23.1
> 60 16.0(12.6 - 20.3) 16.4 ± 3.9 16.9 11.6-23.4
 78 £
The ratio's ofrT 3 /T 3 ,rT 3 /T 4 and T 3 /T 4 were also log-normally distri- -,|£,
buted. No differences were found in the test results between the 25 |£
fasting and the 38 non-fasting subjects. fe,
2. Effect of sex. No differences were found between the values for women \ ft
and for men (table IE. 2). However, a tendency to somewhat higher ; , ||
values of plasma iodothyronines in men was noted. ' §v
3. Effect of age. No differences were found between the values in differ- ; ||
ent age groups (table III. 3). However, a tendency to lower plasma T 3 f|
and higher plasma rT3 concentrations was observed in subjects of 60 j §3
years and older. |y
4. Effect of weight. No correlation was found between the weight and the s |;
plasma concentrations of rT 3 , T 3 and T 4 respectively. |
5. Effect of season. There is an uneven distribution of blood sampling over !
the four seasons (table III. 1), since the possibility of seasonal variation ;
was not appreciated at the outset of the study. Therefore seasonal varia- |
tion in hormone levels was not further investigated, although plasma j |
T 3 and T 4 concentrations in summertime were lower than in the i
winter.
6. Interrelationships of hormone concentrations. Basal TSH concentra-
tion was not related to either rT 3 , T 3 , T 4 or FT 4 index. There was no ••
effect of sex and age on the ratio's of plasma rT3/*T3, rT 3 /T 4 and T 3 /
T 4 , although a tendency to higher rT 3 /T 3 and rT 3 /T 4 and to lower | rl
T 3 /T 4 ratio's in old age was observed. r,

b. TRH-test.
1. Side effects. The observed side effects of a rapid intravenous injection
of 200 jig TRH are listed in table III. 4. The side effects are experienced
within seconds to a few minutes after the TRH injection, and have dis-
rate///-*
Nature and frequency of side effects of 200 fig TRH i.v. as a bolus injection in 63 normal
adults.

no side effects 13
urinary urgency 38
nausea, epigastric discomfort 25
strange, bitter or metallic taste 14
dizziness 12
feeling of flushing 12
headache 4
oppressive feeling in neck or chest 3
heavy feeling in legs 2
palpitations 1
agitation 1
Table III-S
The response of TSH, rT,, T,, and T4 to 200 jig TRH i.v. and to 3 ml. physiological saline Lv. in 6 normal adults.

minutes: -15 0 20 60 120 180 240

TSH mU/1 :TRH 2.2 ± 1.3 2.2 ± 1.5 10.7 ± 3.3 8.4 ± 3.4 5.0 ±2.3 3.9 ± 2.0 2.7 ± 1.5
saline i.7 ± 1.4 1.7 ± 1.1 1.7 ± 1.2 1.5 ± 1.0 1.4 ±1.1 1.4 ± 1.3 1.4 ± 1.1
rT,nmol/l :TRH 0.26 ± 0.06 0.23 ± 0.07 0.22 ± 0.07 0.25 ± 0.06 0.31 ±0.08 0.31 ± 0.07 0.31 ± 0.07
saline 0.23 ± 0.04 0.25 1 0.05 0.25 ± 0.04 0.25 ±0.04 0.27 ±a.O4 0.28 ± 0.05 0.33 ± 0.07
T3nmol/1 :TRH 1.91 ± 0.41 1.89 ± 0.29 1.84i 0.27 2.34 ± 0.76 2.57 ±0.41 2.52 ± 0.51 2.56 ± 0.43
saline 1.681 0.42 1.66 ± 0.44 1.66 ± 0.38 1.65± 0.44 1.73 ±-0.42 1.76 ± 0.52 1.75 ± 0.47
T4 nmol/1 :TRH 104 ± 22 109 ± 24 126 ± 29
saline 1001 19 99 i 16 104 ± 22

,ti5 M^f-K*-'-1'
 80
Table III-6
Effect of sex and age on the TSH response to TRH ( 200 Mg i-v.) in 63 normal adults
(values in mU/1).
geometric mean me- range inner limits
M
yf-j-• -

( x - S D , x + SD) dian P2.S and P97.5

basal TSH : 9 0.7 (<0.3- 2.3) 1.1 < 0 . 3 - 4.3

1.0 < 0 . 3 -
<5
all
0.7 ( < 0 . 3 - 2.3)
0.7 ( < 0 . 3 - 2.3) 1.1 < 0 . 3 -
4.5
4.5 <0.3- 3.1 if;.
peak TSH : 9 .9.7 ( 5.1 - 18.6) 11.4 0.8- 23.0 5 . 0 - 22.0
6 6.4 ( 3.0 - 13.7) 7.0 0.8- 24.3 2 . 8 - 15.5
all 7.9 ( 3 . 8 - 16.5) 8.5 0.8- 24.3 2 . 8 - 22.0
ATSH : 9 8.7 ( 4.5 - 16.8) 9.8 0 . 6 5 - 20.4 4 . 7 - 19.4
a 5.5 ( 2 . 6 - 11.6) 5.5 0 . 6 5 - 19.8 2 . 3 - 13.0
all 6.9 ( 3.3 - 14.5) 7.85 0.65- 20.4 2 . 3 - 19.4

peak TSH :
20 - 2 9 yr. 11.0( 7.3- 16.7) 11.0 6.3 - 2 2 . 0
30 - 3 9 6.8( 4.0- 11.6) 6.7 3 . 0 - 14.0
: 40 - 4 9 10.9( 6.4- 18.4) 13.05 4.« - 2 4 . 3
50 - 5 9 8.9( 4.7- 16.8) 9.9 2.8 - 2 3 . 0
> 60 3.5( 1.3- 9.5) 6.05 0.8* - 8.5
ATSH :
: 20 - 2 9 yr. 10.2( 6.6- 15.6) 10.6 5.3 - 2 0 . 4
30 - 3 9 6.2( 3.8- 10.2) 6.5 2 . 7 - 13.9
40 - 4 9 9.3( 5.6- 15.3) 10.0 4.3 -19.8
50 -59 7.7( 4.0- 14.9) 9.3 2.5 - 2 0 . 3
!
\ > 60 2.8( 1.0- 7.7) 4.8 0.65 - 7.9

appeared nearly always after ten minutes. No serious side effects were
encountered.
2. Control tests. There is no change in plasma levels of TSH, T 3 and T 4 af-
ter administration of physiological saline, in contrast to the increase in
these hormone levels after injection of TRH (table III.5). An increase of
plasma rT3 was observed both after saline and after TRH injection.
Plasma rT3 also increased in non-fasting subjects after TRH.
3. TSH-response. After injection of TRH plasma TSH increased in all
subjects. The peak TSH was observed at 20 minutes in 58 subjects, and
at 60 minutes in S subjects. Peak TSH values at 60 minutes were never
more than 0.3 mU/1 above the 20 min. value. The TSH response to
TRH in women is greater than in men (p < 0.05; table III. 6), and
therefore normal response ranges are given separately for both sexes
(figure HI. 2). The TSH response is not related to basal T 3 or T 4 values.
Basal and peak TSH values are correlated (figure HI. 3); the correlation-
regression equations are: I
 81

1 20

16

j 12

- 8

-15 0 20 60 120 180 240 -15 0 20 60 120 180 240

min min
Figure III. 2. The TSH response to 200 fig TRH i.v. in 32 female and in 31 male normal
adults. Median values arc presented (solid lines), and the inner limits for the percentiles
P2.5 and P97.5 (interrupted lines).

32 females: y = 3.05 x + 7.65 (r =0.57;p < 0.001)

31 males: y = 3.75 x +3:44 (r =0.82; p < 0.001)
63 adults: y = 3.42 x +5.55 (r =0.66; p < 0.001)
The TSH response to TRH is lower in subjects of 60 years and older (p
< 0.02; table III. 6). Peak TSH values of < 1.0 mu/1 were observed in
one female and two males of old age.
4. 7^-response. Plasma T 3 concentrations increased in all subjects after
TRH injection. The peak value is reached at 20 minutes in 1 subject, at
60'in 1 subject, at 120'in 26 subjects, at 180'in 27 subjects and at
240' in 8 subjects. No effect of sex or age on the T 3 response to TRH
was found (table III. 7). Basal T 3 values are correlated both with peak
T 3 values and with the relative increments of T 3 (figure III. 4), but not
with the absolute increments of T 3 . The correlation-regression equations
are:
basalT3 -peakT 3 : y = 0.82x+ 1.22 (r =0.77;p<0.001)
basal T 3 - AT3 absolute : y = - 0.18 x + 1.22 (r =0.22; N.S.)
basal T 3 - AT3 relative : y = - 42 x + 128.52 (r = 0.61; p < 0.001)
The median T 3 response to TRH and the inner limits for the percentiles
P2.5 and P97.5 are depicted in figure III. 5. The inner limit for the per-
centile P2.5 is 0.54 nmol/1 for the absolute increase in T 3 , and 28% for
the relative increase in T 3 . There is an almost complete overlap between
these two criteria, i.e. the subjects with an absolute increase of < 0.54
 82
25

I
20
I
ti
1R

1
IS

10

3
MD 0.3 0.5 1.0 1.5 2X) 25 3.0 35 4O 45
BASAL TSH mU/l 1
Figure III. 3. The correlation between basal TSH and peak TSH after TRH (200 *tg Lv.)
in 63 normal adults (y pe ak TSH = 3.42xbasalTSH + 5.55;r= 0.66; p<0.001).

nmol/1 T 3 all except one had a relative increase of < 28%. The lower
limit for the normal response of T 3 to TRH was found to be an abso-
lute increment of > 0.55 nmol/1, and a relative increment of>30%.The
lowest responses observed are an absolute increase of 0.43 nmol/1 T 3 ,
and a relative increase of 21%. The three subjects with peakTSH values
of < 1.0 mU/1 all had an absolute T 3 increase of > 0.55 nmol/1 and a
relative T 3 increase of > 30%.
5. T4-response. No effect of sex or age on the T 4 response to TRH was
found (table UI.8), although a tendency to a lower response in old age
was observed. Basal T 4 values are correlated to the peakT4 values and
to the absolute increments, but not to the relative increments of T 4 .
The correlation-regression equations are:' -
basal T 4 - peak T 4 : y = 1.18 x-620 (r = 0.91; p<0.001)
basal T 4 - AT4absolute : y = 0.18 x - 0.20 (r = 0.32; p < 0.02 )
basal T 4 - AT4relative : y = 0.03 x +14.73 (r = 0.06; N.S.).
 Table III-7
Effect of sex and age on the T, response to TRH (200 ng Lv.) in 63 normal adults (values as mean ± SD, range between brackets).

; basal T, peakT, AT, AT,

hi (nmol/1) (nmol/1) (nmoj/l) (%) \
i , — . 5
* 9 1.78 ±0.37 2.60 ±0.34 0.82*0.27(0.43-1.60) 50(21-160) co !'
6 1.91 ±0.35 2.87 it 0.44 0.96 * 0.30(0.54 - 2.11) 52(28-140) w J
all 1.85 ± 0.37 2.73 ± 0.41 0.89 ± 0.27(0.43 - 2.11) 51(21 - 160)

20-29yr 1.99 ±0.40 2.88 ±0.49 0.89 ± 0.29(0.43 -1.31) 46(21-69) '
30-39 1.81 ±0.31 2.64 ±0.35 0.83 ± 0.30(0.48 -1.45) 48(21-112)
40 - 49 1.82 ± 0.43 2.81 ± 0.44 0.99 ± 0.38(0.63 - 2.11) 60(28 - 160)
50 -59 1.99 ± 0.24 2.81 ± 0.32 0.81 ± 0.14(0.61 -1.10) 41(33 - 55 ) \f .
> 60 1.64 ± 0.32 2.50 ± 0.40 0.86 ± 0.23(0.54 - 1.28) 55(33 - 105)
 84
•wo

120

100 i

80

60

• $

40

• • ' : •
i
20

1.0 1.2 14 1.6 1.8 20 22 24 2.6 23

BASAL T3 nmol/l

Figure III. 4. The correlation between basal T, and the relative increment of T 3 after
TRH (200 Mg i.v.) in 63 normal adults (y A x % " 4 2 x b a s a l T + 128.52; r=0.61; p<
0.001).

The inner limit for the percentile P2.5 of the T 4 response is 3 nmol/1 for
the absolute increase in T 4 , and 4% for the relative increase in T 4 . The I
increment was less than S nmol/1 in five subjects, and less than 10
nmol/1 in three other subjects. I
c. Cord blood.
The results of the thyroid function tests in 19 cord blood samples are
presented in table III. 9. The values of TSH, rT3 and T 4 are higher, but T 3
values are lower than in the normal adults, and consequently the ratio's of
rT3/T3 and rT3/T4 are increased and of T 3 /T 4 decreased relative to the
values in nonnal adults.
 85

220

2.9 190

-2S _160

130

1.7 100

70
-15 0 20 60 120 180 240 -15 0 20 60 120 180 240
min min
Figure III. S. The T s and T 4 response to 200 ng TRH i.v. in 63 normal adults. Median
values are presented (solid lines), and the inner limits for the percentiles P2.S and P97.S
(interrupted lines).

Table 7/7-8
Effect of sex and age on the T4 response to TRH (200 MS i.v.) in 63 normal adults
(values as mean ± SD, range between brackets).

basal T 4 peakT 4 AT 4
4
(nmol/1) (nmol/1)
(nmol/1)
9 100 ±18 118 ± 2 4 18(-8 - 4 2 ) 1 8 ( - 7 -41)
6 104 ± 17 122 ± 21 .. 18( 2.5 - 32.5) 17( 2 - 29)
all 102 ± 1 7 120 ± 2 2 18(-8 - 4 2 ) 18(-7 - 4 1 )

20 - 29 yr 102 ± 20 125 ± 25 23( 10.5 - 4 2 ) 23( 13 - 3 9 )

30-39 105 ± 14 128 ± 21 22(-8 - 37.5) 21(-7 - 3 8 )
40-49 98 ±20 115 ± 24 17( 3.5 - 4 2 ) 17( 6 - 4 1 }
50-59 103 ± 14 118 ±17 14( 1 - 2 8 ) 14( 1 - 3 1 )
> 60 103 ± 19 118 ± 24 15( 2.5 - 2 8 ) 14( 2 - 2 4 )
Table III-9
Thyroid function tests in 19 cord blood samples from healthy newborns.

geometric mean x± SD median range

( x - S D , x + SD)
TSH mU/1 9.2 ( 5.1 - 16.8 ) 10.6 ± 5.2 10.8 2.3 - 23.0
rT3 nmol/1 2.98 ( 2 . 2 4 - 3.98) 3.11 ± 0.92 2.80 1.95- 5.40
T3mno!/1 1.03 ( 0 . 7 3 - 1.46) 1.09 ± 0.38 1.10 0.50- 2.05
T4 nmol/1 137 ( 113 - 166 ) 139 ± 24 140 85 - 180
TaU 0.86 ( 0 . 8 0 - 0.92) 0.86 ± 0.06 0.86 0.79- 1.09
FT4 index 118 ( 100 - 138 ) 119 ± 18 125 77 - 153
rT,/T 3 xl0- s 2887 (2063 - 4041 ) 3047 ± 1045 2792 1693 - 5200
rT,/T 4 xl0- 3 21.82 ( 17.82 - 26.72) 22.25 ± 4.56 22.76 15.60 - 33.75
T,/T 4 xl0- 5 7.54 ( 5 . 6 3 - 10.11) 7.85 ± 2.29 7.59 4 . 0 0 - 14.14
 87
I
rv III.4. Discussion.
*;.: 4a. Thyroid function tests: basal values.
f:; The plasma concentration of iodothyronines in the normal adults are not
': normally distributed, but have a log-normal distribution. Consequently, the
1 geometric mean was calculated for each test including plasma TSH (the TSH
[ concentrations are also non-parametrically distributed). The arithmetic mean
| is also given, but only to facilitate comparison with the figures in the litera-
I; ture which are nearly always reported as the arithmetic mean (table II. 5, II.
1 14, II. 16 and II. 18). Nevertheless, no great difference is seen between the
geometric andarithmetic means of the plasma concentrations of iodothyronines
(in contrast to the calculated ratio's), suggesting a near normal distribution.
Indeed a normal distribution of plasma iodothyronines has been demonstrated
by Evered et al (1978) in a very large sample (2779 subjects were investigated)
of the population. It should be realized that the normal limits are warning
limits, i.e. at least 2.5% of the values in a normal population is outside each
of the two percentiles. Furthermore, the normal values were obtained from
blood samples taken without venous stasis; in practice, a considerable in-
crease in the iodothyronine plasma concentration sometimes occurs because of
a difficult venapuncture (chapter U.S.).
No effect of sex on the basal values of the thyroid function tests is ob-
served, and this is in agreement with the literature (Ormstcn et al 1971;
Mitsuma et al 1971; Snyder and Utiger 1972; Sanchez-Franco et al 1973;
Meinhold and Wenzel 1974; Birk Lauridsen et al 1974; Blichert-Toft et al
1975; Stepanas et al 1977); only Wenzel et al (1974) report higher TSH
values in young females than in males. The absence of a sex difference in man
is in contrast to the findings of some studies in animals. Female rats have
lower plasma T 3 and higher plasma T 4 values than males. In this respect it is
!. of considerable interest that the peripheral conversion of T4 into T 3 , as
studied very recently by Harris et al (1979) in fresh liver homogenates, is
\. much lower in adult female rats than in males. This difference is caused by a
;• decreased 5 -deiodinating enzyme concentration in the females rather than a
j decreased concentration: of co-factoi(s), since addition of dithiothreitol had
{• no effect on the sex difference. The sex difference in the peripheral conver-
| sion of T 4 is most probably related to sex steroids: the difference is abolished
|; by prepubertal castration and also after peripubertal castration. Testosteron
| administration to castrated male and female rats in vivo increases the conver-
I sion of T 4 into T 3 , whereas 0-oestradiol decreases the T4->T3 conversion in
I the castrated male (although no effect in vitro of estrogens is observed in the
• castrated female rat). At the present time there is no information on a possi-
L We sex-related difference in the peripheral conversion of T 4 in man.
| No effect of age on the basal values of the thyroid function tests could be
| demonstrated in the adult normal population, although a tendency to lower
 88

plasma T 3 values in old age was observed. Low T 3 and high rT3 plasma con-
centrations in old people have frequently been reported (Snyder and Utiger
1972; Rubenstein et al 1973; Herrmann et al 1974; Burger et al 1975; Mol-
holm Hansen et al 1975; Hesch et al 1976; Westgren et al 1976; Nicod et al
1976). The explanation has usually been that there is a change in the peri-
pheral conversion of T 4 with increasing age (Wenzel and Horn 1976). How- •
ever, in an elegant study Olsen et al (1978) demonstrated that low T 3 and ;
high rT3 plasma levels in old age are an effect of disease and not of age. They
found no difference in T 4 , T 3 and rT3 values between healthy young volun- :
teers (aged 18-29 years) and carefully selected healthy elderly people (aged
70-90 years) living at home, but T 3 levels were lower and rT3 levels were
slightly, but not significantly, higher in elderly people living at a nursing
home. The inference is clear: elderly subjects living at home seem to be
healthier than those institutionalized in an home for the elderly. Recently,
Smeulers et al (1979) reported a decrease in T 3 with advancing age, while
levels of T 4 , rT3 and TSH remained unchanged; they found no difference in
serum T 3 between elderly people living at home or at an elderly people
centre. The problem whether impaired T4-*T3 conversion in old age is to be •
regarded as a physiological or a pathological phenomenon, is akin to the
problem of classification of an elevated blood pressure or an impaired glucose '.
tolerance in elderly subjects. It may be concluded that the healthier and j
the more active an elderly subject is, the more likely he is to have plasma ;
iodothyronine values in the same range as young adults. The debate about
the physiological or pathological nature of an impaired conversion of T 4 into
T 3 in old age, is largely semantical and thereby philosophical of character.

In our study body weight and plasma concentrations of iodothyronines are

not correlated. However, a positive correlation between body weight and ;
plasma T 3 (but not with plasma T 4 ) is described by Bray et al (1976). How-
ever, in this study grossly obese subjects were included, in contrast to our •!
control subjects.

The effect of circadian and circannual rhythms on the plasma levels of

' thyrotropin and thyroid hormones cannot be evaluated in our study, since
I blood sampling for basal values was always performed between 08.30 and !
; 10.30 a.m. and seasonal variation was not studied prospectively. A circadian
rhythm of TSH secretion in man is now firmly established, with the highest |
:
levels during the night and a gradual decline to a nadir around 11.00 a.m.
(Nicoloff et al 1970; Patel et al 1972; Weeke 1973; Azukizawa et al 1976; |
- Lucke et al 1977; Chan et al 1978). Plasma-TSH begins to rise in the evening j
i before the onset of sleep, and in fact evidence for an inhibitory influence of '
| sleep has been found (Parker et al 1976). The neuroendocrine mechanisms <
I underlying the circadian TSH rhythm are unknown; they are not related to a '•:
 89
reduced dopaminergic inhibition of the TSH secretion at night (Scanlon et al
1979).
The synchronous variation in TSH and free thyroid hormones excludes
feedback control by thyroid hormones as a cause of the circadian TSH
variation as concluded by Weeke and Gundersen (1978). These authors
describe two rhythms for TSH as well as for FT 3 and FT 4 in man: syn-
chronous diurnal rhythms with lower levels in the day-time and higher levels
at night superimposed by short-term variations with a cyclelength of about 30
minutes; these data suggest a pulsatile release of hormones from the
thyroid gland governed by a pulsatile TSH secretion, although other explana-
tions (e.g. rapid changes of the distribution space of intracellular thyroid hor-
mones) are equally possible. The diurnal variation in the plasma concentra-
tions of total thyroxine and total triiodothyronine is non-synchronous with
the TSH rhythm (De Costre et al 1971; O'Connor et al 1974; Balsam et al
1975; Azukizawa et al 1976; Lucke et al 1977; Chan et al 1978; Weeke and
Gundersen 1978). This pattern is best explained by changes in hormonal
binding by plasma proteins caused by haemodilution and haemoconcentra-
tion due to postural changes (Judd et al 1975) (cf. chapter II. 5). Although of
considerable physiological interest, the fluctuations in plasma hormone con-
centrations due to diurnal variation are minor, and consequently these changes
are of little relevance in routine thyroid testing. In contrast, the variation in
plasma concentrations of T 3 and T 4 due to circannual rhythms is much
greater. Smals et al (1977) describe a seasonal variation in circulating T 3 and
T 4 levels in 13 healthy male adults in the Netherlands. Lowest T 4 and T 3
levels were found in the summer, the mean values being appr. 13 nmol/1 resp.
0.23 nmol/1 lower than in winter (in contrast to a study of Postmes et al
(1974) who could not demonstrate a seasonal variation). The findings of
Smals et al however are in agreement with other reported studies in man. Ex-
posure to the Antarctic cold results in a rise of serum T 3 and T 4 with a return
to normal within 2 days of return to a normal environment (Eastman et al
1974). Although serum T 4 , T 3 and TSH did not change throughout the year
in a subtropical region, the mean urinary T 3 and T 4 excretion (reflecting the
free plasma concentrations of these hormones) were higher during the coldest
months than during the hottest months (Rastogi and Sawhney 1976). No
seasonal variation was found in university employees who lived in air-con-
ditioned rooms, but plasma T 3 concentration increased significantly in winter
(T 4 and TSH did not change) in adult men living in a cold environment
(Nagata et al 1976). The changes in plasma thyroid hormone concentrations
in response to cold exposure are probably TSH mediated (Tumisto et al
1976), although there is no unanimous opinion on this point. Nevertheless,
cold exposure in the rat stimulates the release of TSH, which can be suppressed
by pretreatment with antibodies to TRH (Mori et a!1978) indicating a physio-
logical role for TRH in the TSH response to cold. A shift in the conversion of
 90
T 4 towards a preferential generation of T 3 might be another mechanism in
the adaptation to cold, but so far this has not been studied.

4b. The TRH test.

it' The side effects of an intravenous injection of TRH occur within
seconds to minutes, and prior to the rise in TSH; the side effects are there-
fore not TSH mediated. It has been demonstrated that TRH is also
present in the mammalian brain outside the hypothalamus (Wilber et al
1976), and its presence in the digestive tract of the rat has also been reported
(Morley et al 1977). Recent studies indicate that TRH has a neurotransmittor
function in the central nervous system outside the hypothalmus (Guillemin
1977). In this respect it is of interest that the urge to micturate in response to
TRH was also observed in a patient with complete spinal cord transection at
the level of the eight thoracic vertebra (Rosenthal 1974), and that a decrease
in gastric motility after TRH has been seen endoscopically in human subjects
(Dolva et al 1978). Thus an action of TRH as a neurotransmittor either cen-
trally or locally in the smooth muscle of the gastrointestinal and genitourinary
tracts may explain most of the side effects (cf. Ormston et al 1971). Exten-
sive clinical experience over the last eight years has proven that the TRH test
is a safe and reliable procedure.

In our study, we rather arbitrarily selected a dosage of 200 /ig TRH. As

judged from dose-response curves 200 jig TRH i.v. elicits a submaximal
response of TSH, and maximal responses are obtained at dosages of 400-500
Mg TRH i.v. (Haigler et al 1971; Snyder and Utiger 1972;Otsuki et al 1973).
Other dosages and/or routes of administration (intramuscularly, orally) have
been recommended in the literature with or without measurement of theT 3
and T 4 responses (Haigler et al 1972; Noel et al 1974; Azizi et al 1975; Van
Kersen et al 1975; Staub et al 1976; Bremner et al 1977; Vogt et al 1978). We
have sofar not been convinced of the advantages claimed in these studies,
especially since in our hands the T 3 response to TRH proved to be a very
reliable criterion. Therefore we continue to use 200 /utg TRH intravenously,
in accordance with the majority of the studies reported in the literature.

As judged from the control tests, a significant increase in plasma values

of TSH, T 3 and T 4 is observed after TRH but not after physiological saline.
No difference between TRH and saline is found in the plasma levels of rT3.
The slight increase in plasma rT3 during both tests is not due to fasting, since
the same finding is reported by Bremner et al (1977) in non-fasting normal
subjects. Reverse T 3 is present in the human thyroid gland (Chopra 1974;
Burman et al 1977) and is secreted into blood especially when the gland is
stimulated with TSH (Chopra et al 1975; Westgren et al 1976; Hooper et al
1978), but clearly intensive TSH stimulation is needed to obtain a measurable
 91
response. Burman et al (1974) report a mean increase of 0.04 nmol/1 after
r 500 ng TRH i.v. in 10 normal human subjects and claim that this increase is
significant, but no control tests were performed.
We did not study the rT3 response any further, no explanation can be
offered for the rise of rT3. Also, the T4 response to TRH was not studied
further, since 8 of the 63 normal adults showed an absolute increment of
less than 10 nmol/1 implying that the T 4 response discriminates poorly be-
tween the euthyroid and the hyper- or hypothyroid state. A similar minor T 4
response to 100-500 /ig TRH i.v. peaking around 4-6 hrs after TRH has been
common experience (Hollander et al 1972; Lawton et al 1973; Uller et al
1973). In case no TSH or T 3 assay is available and one has to rely on T 4
determinations only, higher doses of TRH are needed (Lawton et al 1973;
Azizi et al 1975; Van Kersen et al 1975; Vogt et al 1978).

Although peak TSH was < 1.0 mU/1 in three elderly subjects, a measurable
TSH response to TRHv/as observed in all 63 normal subjects. This is in agree-
ment with Andersen et al (1971), and with Birk Lauridsen et al (1974) who
found a rise in TSH of < 2.0 mU/1 after TRH in 2 out of 60 controls. It is
likely that no more infonnation is obtained by extending the measurement of
TSH beyond 20 minutes. Also, the use of A TSH instead of the peak TSH
fl value has no clear advantage (Sawin and Hershman 1976). Sometimes the
TSH value at 60 minutes is somewhat higher than the 20 min. value, and this
pattern seems to be a normal variant (Birk Lauridsen et al 1974; Sawin and
Hershman 1976). We found the peak TSH to be correlated with basal TSH,
indicating a proportional magnification of basal TSH by TRH; this is also
described by Weeke (1974), Birk Lauridsen et al (1975) and Sawin and Hersh-
man (1976). We, nor Wenzel et al (1974) could relate the peak TSH values
to basal T 3 or T 4 , but Sawin and Hershman (1976) report a moderate nega-
tive correlation of peak TSH with basal T 4 (the coefficient of correlation be-
tween peak TSH and basal T 3 or T 4 in our study is indeed higher for T 4 than
T 3 , but failed to reach statistical significance in both). This would indicate
that plasma T 4 is one of the determinants for the secretion of TSH which is
in agreement with the findings in hypothyroid patients (see chapter IV) and
the interesting studies of Larsen and co-workers (Silva and Larsen 1977; see
further chapter V. 4).
Our results indicate that the TSH response is greater in females than in
males. This has been reported by many others (Bowers et al 1971; Ormston et
al 1971; Haigler et al 1971; Sanchez-Franco et al 1972; Noel et al 1974; Wen-
zel et al 1974; McNeilly et al 1974; Blichert-Toft et al 1975; Sawin et al
1978); in the studies who fail to show this sex difference, the tendency to a
greater response in women is nearly always observed (Snyder and Utiger 5
'•3.
1972; Weeke 1974; Birk Lauridsen et al 1974; Vogt et al 1978). The sex
difference cannot be caused by testing at different times of the menstrual
 92
cycle, since the TSH response to TRH is the same at the follicular phase and
at the luteal phase (Weeke 1972; McNeilly et al 1974; Sawin et al 1978),
despite the initial findings of Sanchez-Franco et al (1972) describing a greater
TSH response before ovulation than afterwards. Nor can the greater increase
in TSH in women be explained by a relatively higher dose of TRH adminis-
tered to women because of their lower body weight, since in subjects matched
for height and weight women still demonstrated a tendency to higher TSH
responses than men: peak values 16.8 vs. 14.3 mU/1 (Snyder and Utiger 1972).
The common explanation has been that the greater TSH response in females
is due to higher levels of plasma estrogens. The absence of a difference
between the TSH responses in elderly women and men favours this explana-
tion (Wenzel et al 1974) although this finding has been disputed (Snyder
and Utiger 1972). The enhanced TSH response seen in males after the ad-
ministration of estrogens also favours a causative role of estrogens (Faglia
et al 1973; Mortimer et al 1974; Smyth et al 1977) although again this
has not been a universal finding (Woolf et al 1973; Carlson et al 1973).
Sawin et al (1978) conclude that the higher average estradiol levels in nor-
mally menstruating women tend to enhance the TSH response to TRH,
but that cyclical changes in estradiol and progesterone have no effect.
The mechanism by which estrogens enhance the TSH response to TRH
is unclear; in this respect it is of interest that preincubation of rat anterior
pituitary cells with estradiol led to increased TSH responsiveness to TRH
(Labrie et al 1978), that estrogens decrease the metabolic clearance rate of
TSH in the laboratory animal (Sawhney et al 1978), that the thyroid gland
in normal females is larger than in normal males (Boyd 1961), and that the
peripheral conversion of T4-*T3 in female rats is lower than in males (Harris
et al 1979).
Similarly, conflicting results are reported on the TSH response to TRH as
a function of age. Snyder and Utiger (1972) and Birk Lauridsen et al (1974)
find no effect of age in females, but a decrease in elderly males. In contrast,
Wenzel et al (1974) report a lower response in elderly females but not in
males, and Sakoda et al (1976) finds a decrease when applying 400 MgTRH
i.v. but not at doses of 50-100 jug. Again, the general finding is that the lowest
responses are found in elderly subjects (Otsuki et al 1973; Cuttelod et al
1974; Blichert-Toft et al 1975) like in our study. Some authors conclude that
it is necessary to determine normal limits of the TSH response to TRH for
males and females seperately, and also for each decade (Snyder and Utiger
1972; Wenzel et al 1974). We are of the opinion that, given the barely
significant or insignificant differences between males and females and be-
tween young and old people, it is justified to apply one normal range for the
whole population. Common sense then dictates that a low TSH response in a
young female is more likely to be abnormal than in an octagenarian.
 93
The T3 response to TRH in our study is not influenced by sex or age, in
accordance with studies of Wenzel et al(1974) and Vogt et al (1978). In fact,
the subjects with peak TSH values of < 1 mU/1 all had a clear increase in T 3
levels; thus measuring the T 3 response to TRH can be advantageous. In this
respect we disagree with other authors who find the T 3 increase too small or
too inconsistent to be of diagnostic value (Wenzel et al 1974; Vogt et al
1978). The reason for this is no doubt that, whereas others have only deter-
mined T 3 at 120 or 180 minutes, we measure the T 3 response for 4 hours,
and it has been clearly documented that plasma T 3 peaks after 2-4 hours
(Shenkman et al 1972; Lawton et al 1973; Uller et al 1973). The fact that
the maximal increase in T 4 is seen 4-6 hours after 200 jug TRH i.v. (Law-
ton et al 1973; Uller et al 1973) again illustrates the preferential secretion
of T 3 over T 4 in response to TSH stimulation.

4c. Cord blood.

The results of the determination of hormone levels in the cord blood
samples are most remarkable (very high rT3 and low T 3 levels, in combina-
tion with elevated T 4 and TSH levels), and are in agreement with previous
I-
reports (Larsen 1972; Chopra et al 1973, 1975; Erenberg et al 1974). The
findings strongly suggest that impairment of the peripheral conversion of T 4
is involved; indeed Chopra et al (1975) conclude from kinetic studies in fetal
and adult sheep that peripheral production of T 3 from T 4 is reduced in the
fetus, while the rate of conversion of T 4 to rT 3 is comparable to that in the
adult; the very high rT 3 concentrations are the result of an increased produc-
tion of the substrate T 4 , and of a lower metabolic clearance rate as compared
to adult sheeps. The relevance of these findings are discussed in chapter DC.
The high TSH values are explained by thesuddenTSH surge occurring at birth
as a result of cooling; the plasma TSH concentration in the neonate reaches a
maximum level within 30 min of delivery, and decreases thereafter.

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5
I
CHAPTER IV.

STUDIES IN PATIENTS WITH THYROID DISEASES

¥.'
IV.I. Introduction.

Thyroid function was evaluated in a series of patients referred to our

clinic. The aim of this study is to obtain a spectrum of thyroid function
disorders ranging from severe hyperthyroidism to severe hypothyroidism,
and to evaluate the usefullness of the various tests in the diagnosis of
increased or decreased thyroid function as an indicator of thyroid disease.

IV.2. Patients and methods.

Patients who for the first time were referred to our clinic under the
suspicion of thyroid disease, entered the study. Patients who had been treat-
ed with thyroid and / or antithyrpid drugs in the six months before the date of
referral, and patients with hypothalamic-pituitary disorders were
excluded. Patients medicated with beta-adrenoceptor blocking agents, or
with drugs which influence thyroid hormone binding to the plasma
proteins, and patients with co-existent acute or chronic non-thyroidal
diseases were not excluded. The nature of the thyroid disease was
determined by clinical examination, by the presence or absence of thyrc A
antibodies, and by thyroid scintigraphy. Ophthalmic Graves' disease is the
term we used to describe patients with the ophthalmic manifestations of
Graves' disease in the absence of clinical hyperthyroidism or a past history
of hyperthyroidism (Hall et al 1970).
Blood sampling was performed through an indwelling venous catheter,
and after obtaining two basal samples 200 Kg TRH was rapidly injected
intravenously (see chapter III. 2). The basal value of each test was
calculated as the mean of the -IS' and 0' samples. In calculating ratio's etc.
SO percent of the detection limit of the assay was taken when the value was
below the detection limit of the assay.
Patients with abnormal thyroid function tests were classified according
to the FT4 index and T3 values. If the FT4 index and the T3 values were both
normal, the TSH-response to TRH was used for further classification.
I
IV.3. Results. I
One hundred forty-eight patients were tested, and abnormal thyroid
 1
Classification of 125 patients with abnormal results of thyroid function tests (t = increased; I = decreased; N = normal; GO >» Graves disease,
s
TA = toxic adenoma, TMG = Toxic multinodular goitre, SAT = subacute thyioiditis, AIT autoimmune thyroiditis, STT = subtotal thyroid-
ectomy, TT = total thyroidectomy).

classification number of pat. age, yrs. weight, kg clinical diagnosis laboratory diagnosis
group FT4 index T, ATSH total (females) (xtSD) (x ± SD)

At t i 55 (43) 53.8 ± 1 6 . 9 62.6 i 12.5 hyperthyroidism due to: overt hyperthyroidism

GD (33)
TA( 9)
TMG (12)
SAT( 1)
B N t j, 11 ( 9) 60.5 ±11.1 60.3 ± 1 1 . 9 hyperthyroidism due to: mild hyperlhyroidism
GD( 4) ("Ts-toxicosis")
TA( 1)
TMG( 3)
ophthalmic GD ( 3)
C N N i 15 (11) 58.5 ± 1 3 . 0 66.5 ± 1 1 . 8 equivocal hyperthyroidism subclinical hyperthy-
due to: roidism
GD( 3)
TA( 3)
TMG( 4)
ophthalmic GD ( S)
D N N t 6 ( 4 ) 53.3 ±19.1 66.1 ± 17.9 equivocal hypothyroidism subclinical hypothy-
due to: roidism
AIT( 2)
STT( 2)
ophthalmic GD( 2)
E i N t 6 ( 6 ) 62.2 ± 12.0 67.11 7.0 hypothyroidism due to: mild hypothyroidism
A1T( 4)
STT( 1)
SAT( 1)
it 27 (21) 54.5 ±16.7 74.7 ± 10.5 hypothyroidism due to: overt hypothyroidism
AIT(18)
STT( 6)
TT ( 3)
G t N I 5 ( 3 ) 66.0 ± 5.1 61.51 10.4 equivocal hypcrthyroidism ("T4-toxicosis")
due to:
GD( 3)
TA( 1)
TMG( t)

K'^! ! HSSc^?sC?55' : rr-'™;' K '-" ; '"' :! "'''''"."•'"~- - •^•*'*? ite ff^» i >Wjsii*^^ -,,...,,. ..,„,,„,
 101
HYPERTHYROIDISM EUTHYROIDISM HYPOTHYROIDISM
§ overt mild subclinical subclinical mild overt
A B C NORMALS D E F
GROUP (n=55) (n=ni ln=15) (n=63) (n=6) (n=6) (n=27)
_ 60

i | 20

<0.3

| 0.4

t 0
_ 4
\
I 2
c
& 0
_ 200
\
§ 100
c
? 0
1.4

& 0.6
240

I 120
£ 0

Figure IV. 1.
Median values of thyroid function tests in 63 normal adults and in 120 patients with thyroid
function disorders (groups A-F, for classification see table IV. I). The horizontal linesdepict
the inner limits for the percentiles P2.5 and P97.5 of the normal values.

function tests were found in 125. These were divided in the groups A-G (see
table IV. 1) Four patients in group A and one in group B were on oral
contraceptive pills, two in group A and one in group G were on B-blockers.
One patient had a normal FT4 index and an increased plasma T3, but also
an increased TSH-response to TRH; therefore she was aUocated to group
D. Median values and the observed range of the thyroid function tests in
 102
the groups A-F are presented in table IV.2 (see also figure IV. 1).
All unequivocally hyperthyroid patients were allocated in the groups A
I,
and B. Of these 66 patients, Graves' disease was diagnosed in 40 (60,5%),
and toxic nodular goitre in 25 (38%). The patients with toxic nodular

Table IV-2
Thyroid function tests in 63 normal adults and in 120 patients with thyroid function dis-
orders (groups A - F , for classification see table IV-1). The median values are presented;
in brackets the range in the groups A — F, and the inner limits for the percentiles P2.5
and P97.5 in the normal subjects.

TSH mU/1 rT3nmol/l T3 nmol/1

group A <0.3 0.67 3.85

(n = 55) « 0.3 - 1.35) ( 0.26-4.05) (2.50 - 13.35)
group B <0.3 0.40 2.70
« 0.3 - 1.1 ) ( 0.22-0.69) (2.53- 3.94)
group C <0.3 0.28 2.00
(n=15) (< 0.3 - 1.1 ) ( 0.17 - 0.57) (1.63- 2.45)
normals 1.1 0.22 1.75
(n = 63) « 0.3 - 3.1 ) ( 0.14-0.38) (1.30- 2.45)
group D 4.9 0.29 2.19
(n = 6) ( 2 . 6 - 46 ) ( 0.18- 0.36) (1.75- 2.62)
group E 17.2 0.13 1.66
(n = 6) ( 5 . 7 - 60 ) ( 0.08 - 0.17) (1.44- 2.15)
group F 61 <0.03 0.88
( 17 - 3 4 3 ) « 0 . 0 3 - 0.11) (0.19- 1.25)

T4nmol/1 T,U FT4 index

group A 193 1.21 219

(n = 55) ( 123-338) (0.81 - 1.66) (123 - 476)
group B 120 0.94 113
( 107-145) (0.59 - 0.99) ( 85 - 122)
group C 102 0.93 88
(n= 15) ( 75 - 135) (0.75 - 1.18) ( 73 - 122)
normals 101 0.93 95
1
(n = 63) ( 75 - 135) (0.81 - 1.07) ( 70-122)
group D 84 0.84 72
( 80 - 107) (0.71 - 0.91) ( 7 0 - 80)
group E 60 0.88 51
( 4 2 - 68) (0.74 - 0.95) ( 37 - 57)
group F 17 0.71 10
« 5 - 53) (0.55 - 0.87) ( 2-44)
 103

I
3
& •
rT3/T3xl0" rT 3 /T 4 xl0- 3 T 3 /T 4 XIO -

group A 170 3.66 22.2 ;

(n=55) (58 - 387) (1.80-14.11) (12.4- 46.0) I
* \

group B 130 3.18 24.1 j

(76 - 218) (1.85- 5.17) (17.8- 36.8) j
group C 142 2.83 20.2 1
;
(n=15) (90 - 285) (2.05- 5.56) (15.5 - 28.5)
normals 121 2.20 18.2 !
(n = 63) (69 - 209) ( 1 . 3 8 - 3.53) (12.8- 23.4) ;
group D 118 3.03 23.9 \
(n = 6) (93 - 195) (2.19- 4.23) ( 2 0 . 3 - 28.9) \
group E 77 2.15 29.9
(n=6) (45 - 111) ( 1 . 9 0 - 3.33) ( 2 3 . 9 - 41.9)
group F 43 2.50 55.8
(n=27) (13 - 219) (0.43 - 16.00) (11.4 - 350. )

goitre are older and have lower plasma T3 and T4 values than the patients
with Graves' disease (table IV.3). First-onset thyrotoxicosis was seen in
47 of 65 patients (72%), and recurrent thyrotoxicosis occurred in 18 (28%):
13 out of 54 patients in group A (24%), and 5 out of 11 patients in group B
(45%). The incidence of Graves' disease and toxic nodular goitre was the
same in patients with or without recurrence (table IV.4). Recurrent
thyrotoxicosis was associated with lower plasma T 3 and T4 values and
with older age in comparison to first-onset thyrotoxicosis. Eleven out of
the 66 patients of the groups A and B had increased plasma T3 levels (and
FT3 index values) but a normal FT 4 index. The incidence of this so-called
"T3-toxicosis" is thus 17%. Plasma T3 values in T3-toxicosis were lower
than in hyperthyroid patients with an increased FT4 index (group A), but
plasma T4 values in T3-toxicosis (although by definition within the normal
range) were higher than in the normal adult population.
All unequivocally hypothyroid patients were allocated to the groups E
and F. The cause of the hypothyroid state in these 33 patients was
autoimmune thyroiditis in 22 (67%), subtotal thyroidectomy in 7 (21%),
total thyroidectomy in 3 (9%) and subacute thyroiditis in 1 (3%).- A normal
plasma T3, but a decreased FT4 index was found in 6 of these patients (18%;
group E). The decrease in FT4 index in these patients is less than in the
patients of group F who also demonstrate decreased plasma T3 values.
In between the hyperthyroid patients of the groups A and B and the
hypothyroid patients of the groups E and F are 21 patients (group C and
D) with normal plasma T3 and FT 4 index values; they were considered
abnormal because of an abnormal TSH response to TRH stimulation.
 104
Table IV-3
I Thyroid function tests (given as geometric mean, between brackets x - SD and x + SD)
in 65 hyperthyroid patients with Graves' disease or nodular goitre.

Graves' disease toxic adenoma toxic multinodular

goitre
¥(•-•
number of
patients 40 10 15
females 29 8 14
males 11 2 1
age yrs ( x ±
SD) 47.8 ± 15.1 64.0 ± 10.7 69.4 ± 8.6

rT3 nmol/1 0.66 ( 0 . 3 9 - 1.13) 0.56( 0.33 - 0.96) 0.68( 0.45 -- 1.03)
T 3 nmol/1 4.27 ( 2.25 - 8.10) 3.53( 2.67 - 4.66) 3.78( 2.86 -- 5.01)
T4nmol/1 186 (138 - 249 ) 161 (127 -205 ) 168 (130 -- 2 1 8 )
T3U 1.15 ( 0.93- 1.42) 1.16( 0.95 - 1.41) 1.13( 0 . 9 8 -- 1.31)
FT 4 index 213 (134 - 338 ) 187 (130 -269 ) 190 (131 -- 2 7 6 )

Fifteen had a decreased or absent TSH response (group C), and six showed
an increased TSH response (group D). The FT 4 index values in the patients
of group D are lower, but plasma T3 values are higher than in the normal
subjects.
All patients of the groups A and G responded favourably to appropriate
treatment. Almost all patients of the groups B and F were also treated, and
the majority improved clinically. Several patients of group C and D were
also treated specifically, however the response to treatment of these
individuals was much less consistent.

Table IV-4
Thyroid function tests (given as geometric mean, between brackets x - SD and x + SD) in
47 patients with fust-onset thyrotoxicosis and in 18 patients with recurrent thyrotoxicosis.

first-onset recurrence

number of patients 47 18
Graves' disease 29 11
toxic adenoma 8 2
toxic multinodular goitre 10 5
age yxs (x + SD) 52.3 ± 17.3 61.4 ± 10.8

rT9 nmol/1 0.66 ( 0.40- 1.10) 0.6K 0.37 - 1.02)

T9 nmol/1 4.40 ( 2.87- 6.74) 3.72( 2.53 - 5.47)
T4 nmol/1 182 (136 - 244 ) 166 (130 - 2 1 2 )
T3U 1.18 ( 0.98- 1.42) 1.07( 0.89 - 1.30)
FT4 index 215 (140 - 329 ) 179 (119 - 2 6 8 )
 105
JF-J
The TSH- and T3-response to 200 Mg TRH lv. in 63 normal adults and in 120 patients
with thyroid function disorders (groups A - F , for classification see table IV-1). The me-
dian values are presented; in brackets the range in the groups A - F , and the inner limits
for the percentiles P2,5 and P97.5 in the normal subjects.

basal TSH mU/1 peak TSH mU/1 ATSHmU/1 ATSH %

1 group A
(n = 55)
<0.3 <0.3 0
(< 0.3 - 1.35) (< 0 . 3 - 1.9) (- 0 . 4 - 1.05)
group B <0.3 <0.3 0
« 0.3 - 1.1 ) « O.i- 1.3) (- 0.13- 0.4 )
group C <0.3 0.5 0.1
(n=15) « 0.3 - 1.1 ) « 0 . 3 - 2.0) ( 0 - 1.85)
normals 1.1 8.5 7.85
(n = 63) « 0.3 - 3.1 ) ( 2 . 8 - 22.0) ( 2 . 3 - 19.4 )
group D 4.9 39.4 34.5 713
(n = 6) ( 2 . 6 - 46 ) ( 25.8-103 )( 19.5- 57.1 ) (124 - 1444)
group E 17.2 62.4 47.5 246
(n = 6) ( 5 . 7 - 60 ) (23-155 ) ( 17.4 - 95 ) (144- 400)
group F 61 136 57 87 §
(n = 27) ( 17 - 3 4 3 ) ( 33 - 7 0 8 )( 10 - 470 ) ( 24 - 555)
$
basal T3nmol/1 peakT3 nmol/1 AT3nmol/l AT,

group A 3.85 4.40 0.30

(n = 55) (2.50- 13.35) (2.96 - 13.60) (-0.35 - 1.31) ( - 4 - 29)
group B 2.70 3.25 0.25 9
(2.53 - 3.94) (2.60- 4.23) (-0.05 - 0.68) ( - 2 - 26)
group C 2.00 2.35 0.37 18
(N=15) (1-63 - 2.45) (1.70- 3.08) (-0.18 - 0.93) ( - 9 - 47)
normals 1.75 2.70 0.83 45
(n = 63) (1.30- 2.45) (2.15- 3.40) ( 0.55-1.31) (30-105)
group D 2.19 2.92 0.66 33
(n = 6) (1.75 - 2.62) (2.30- 3.70) ( 0.43-1.41) ( 1 8 - 77)
group E 1.66 2.00 0.42 26
(n=6) (1.44 - 2.15) (1.26- 3.05) (-0.50 - 0.90) (-28 - 42)
group F 0.88 0.88 0.10 20
(n = 27) (0.19- 1.25) (0.24- 1.52) (-0.15 - 0.55) (-16 - 78)

The TSH- and T3-responses to TRH in the patients of the groups A-F are
presented in table IV.S. All patients of the groups A, B, and C
demonstrated a severely impaired or absent TSH - response. The patients
of the groups D, E, and F demonstrated a greater than normal TSH -
 106
200 r
-I
100
I
50

10

CO normal

0.5

detection limit
0.3

V- = _ = .__ f= . _^c
0.1

I Figure IV. 2.
.15 0 20 60 120 180 240
The TSH-rcsponse to 200 wg TRH i.v. in 63 normal adults and in 120 patients with thyroid
function disorders (groups A-F, for classification see table IV. 1; the median values of each
groups are presented, note the logarithmic scale).
V- 107
HYPERTHYROIDISM EUTHYROIDISM HYPOTHYROIDISM
overt mild subclinical subclinical mild overt
B C NORMALS D E F if
GROUP (n=55) (n=ni (n=15) (n=63) (n=6) (n=6) (n=27)

too

80

60

*40

20

-20

1.4

1.1

_0.8

I 0.5
c

<i0.2

.0.1

-0.4
Figure IV. 3.
The T,-response to 200 wg TRH i.v. (upper panel: relative increment; lower panel: absolute
increment) in 63 normal adults and in 120 patients with thyroid function disorders (groups A-F,
for classification see table IV. 1). The horizontal lines depict the inner limit for the percentile
P2.S of the normal response.

response, i.e. in all subjects the response was above the inner limit for the
percentile P97.S of the TSH - response in normal subjects. The relative
t
increment of TSH after TRH decreased progressively from groups D to F,
as can be seen in fig. IV.2.
The individual values for the absolute and relative increment of T 3 in
response to TRH, are depicted in figure IV. 3. None of the hyperthyroid
1
patients of the groups A and B was observed to have a relative increment
greater than 30%, although 18 patients had an absolute increment greater
 108
than O.SS nmol/1. Two patients of group C, four patients of group D and
three patients of group E had a normal relative and absolute increment of
T3. Seven of the severely hypothyroid patients of group F had a normal
1-'.
relative increment of T3, but only one had a normal absolute increment of
0.55 nmol/1.
r
As judged from the inner limits for the percentiles P2.5 of both the normal
absolute and relative increments of T3 in response to TRH, all
hyperthyroid patients with an increased plasma -T3 and all hypothyroid
i
patients with a decreased plasma -T3 (except one) have an impaired T3-
response.
Five patients with equivocal hyperthyroidism (group G) had elevated
FT4 index values but a normal plasma T3; the TSH- and T3-response to
TRH was impaired in each (table IV. 6); they are considered to have "T4- V'
toxicosis". Their case histories are presented below.

Patient I, a 59-years old female with sickle cell anaemia, was admitted because of
abdominal complaints and a decrease in renal function. A diagnosis was made of
necrotising papillitis; her clinical condition'improvcd without specific treatment. She
was on digoxin for four years because of atrial fibrillation, and had been treated ten
years before admission with carbimazole because of thyrotoxic Graves' disease. Al-
though clinically euthyroid, a TRH test was performed. In the two weeks prior to the
test, oral and intravenous cholecystography and intravenous urography was per-
formed. She was discharged without anttthyroid treatment, and died some months
later from unknown cause.
Patient 2. a 64-years old male schizophrenic, experienced a considerable loss of weight
of 16 kg in three months. He was otherwise healthy, and no goitre was palpable.
Hyperthyroidism was suspected, and thyroid function tests were compatible with the
diagnosis of "T4-toxicosis". However, at the time the results were known, his condition

Table IV-6
Thyroid function tests (basal values, and the TSH- and T3-response to 200 ng TRH Lv.) in
five patients with an elevated FT 4 index but normal T3 values (group G; "T4 -toxicosis").

patl pat. 2 pat. 3 pat. 4 pat. 5 x±SD

TSH mU/1 <0.3 <0.3 <0.3 <0.3 <0.3 <0.3

rT3 nmol/1 0.81 1.13 0.79 0.52 0.39 0.73 ± 0.29
T3 nmol/1 2.11 2.33 1.83 1.73 2.00 2.00 ± 0.24
T4 nmol/1 176 163 150 150 143 156 ± 13
T3U 1.02 1.27 1.27 0.99 1.04 1.12 ± 0.14
FT4 index 180 207 191 149 149 175 ± 26
rT 3 /T 3 xl0- 3 384 485 432 301 195 359 ± 114
rT,/T 4 xl0" 3 4.60 6.93 5.27 3.47 2.73 4.60 ± 1.63
T 3 /T 4 xl0" 3 12.0 14.3 12.2 11.5 14.0 12.8 ± 1.3
ATSH mU/1 0 0 0 1.15 0 0.2 ± 0.5
AT3 nmol/1 -0.37 0.45 0.33 0.25 0.55 0.24 ± 0.36
AT3% -17 19 18 15 28 13 ± 17
 109

240
min

Figure IV. 4.
TheTSH-and T3-responseto200wgTRH i.v. in an elderly female patient while asymptomatic
(solid line), and during an hysterical twilight state (broken line).

had improved and no antithyroid treatment was instituted. Plasma T4 gradually

decreased into the normal range, and has remained normal for two years. During the
observation period the patient was on medication with various psychotropic drugs.
Patient 3, a 73-years old female, was admitted because of fever from unknown origin.
Atrial fibrillation and ischemic heart disease were diagnosed, but no pulmonary
embolism could be demonstrated. She was treated with digoxin, furosemide and
acenocoumarol. Two weeks prior to the TRH test plasma T, was 3.00 nmol/land
plasma T 4 14S nmol/1, one week later oral cholecystography was performed. At the
time of the TRH test plasma T 3 was 1.83 nmol/1 and T 4 ISO nmol/1. Thyroid
scintigraphy demonstrated a small multinodular goitre. The fever did not respond to
antibiotics, but disappeared during entithyroid medication.
Patient 4, a 67-years old male, was referred because of atrial fibrillation with a high
ventricular response, which could not be suppressed satisfactorily with digoxin. The
patient had no other signs or symptoms of thyrotoxicosis, and no goitre was palpable.
A first blood sample revealed increased values of T } (3.1 nmol/1) and T4 (145 nmol/1).
Propranolol was added to the medication, and this had a favourable effect: the
ventricular rate decreased. The TRH test was performed under propranolol
medication.
Patient 5, a 67-years old female, was admitted to the psychiatric c!:.nic because of
melancholy. She was treated with chlorodiazepoxide, perphenazine, amitryptiline and
B-pyridyl-carbinol (Ronicol). Hyperthyroidism was suspected clinically, and thyroid
scintigraphy revealed a toxic adenoma. The patient improved under carbimazole.

IV. 4. Discussion.

4a. The spectrum of thyroid function disorders.

The plasma iodothyronine concentrations found in subjects with
increased, normal or decreased thyroid function, are depicted in figure IV.
I
1. The figure suggests that in the development of the hyperthyroid state the
production of T3 is increased first, to be followed by an increased secretion
 -—-?•"

110
I
yv>
of T4. In this view T3 toxicosis is just a forerunner of the classical
thyrotoxic state in which both T4 and T3 are produced in excess. In
I contrast, in the development of hypothyroidism production of T4 is first of
all decreased, later followed by a decrease in the production of T3. Several
longitudinal studies have indicated that this proposed sequence can indeed
occur (Hollander et al 1971; Patel and Burger 1973; Marsden and
McKerron 1975; Marsden et al 1975; Toft et al 1975).
It is thus plausible that patients with T3-toxicosis are less thyrotoxic
than patients in whom both T3 and T4 are increased, and patients with a
decrease in plasma T4 only have a milder form of hypothyroidism than
patients in whom plasma T3 is also decreased. The relation between the
plasma T3 concentration and the severity of the thyroid function disorder is
supported in the literature (Surks et al 1972). The plasma T3 concentration
in "classical thy rotoxicosis" where plasma T4 is also increased, is higher than
in T3-toxicosis in which a clinically less severe hyperthyroidism is reported
(Ivy et al 1971; Marsden and McKerron 1975). Mild hypothyroidism or
clinical euthyroidism with a decrease in plasma T4 only is described by Patel
and Burger(1973). Evered etal(1973)andStepanasetal(1977). In none of
these studies (including ours) the clinical state has been evaluated quan-
titatively, and consequently the relation between the plasma T3 concentra-
tion and the clinical state cannot be expressed numerically. In general, how-
ever the level of the plasma T3 concentration reflects the severity of the
thyroid function disorder, although exceptions to this rule are not rare.
Figure IV. 1 and the T 3 /T 4 ratio's show that there seems to be an
overproduction of T 3 relative to T4 in both hyperthyroidism and
hypothyroidism, and this has been verified in kinetic studies (Larsen 1972;
Nicoloff et al 1974). The overproduction of T3 can be explained by the
development of iodine deficiency in the thyroid gland, which then leads to
a preferential synthesis of T3. Such a mechanism is Ideologically sound, at
least in the development of hypothyroidism: T 3 is metabolically four times
more active than T4, the preferential synthesis of T3 saves 25% iodine, so
that switching from T4 to T3 synthesis results in an approximately sixfold
increase of the hormonal effect at the same expenditure of iodine (Havard
1974). Clinical and experimental studies support this explanation (Greer et
al 1968; Pineda etal 1970). The increased incidence of T3-toxicosis in areas
with endemic goitre also favours such a pathogenesis (Hollander et al
1972). The increased TSH stimulation in hypothyroidism may be
responsible for a preferential secretion of T3 by the thyroid gland, in order
to maintain euthyroidism (Sterling 1970; Patel and Burger 1973; Evered et
al 1973). In this respect it is of interest that plasma T3 values in the patients
with subclinical hypothyroidism (group D) are higher than the average
plasma T3 values of the normal subjects, and in fact an abnormally
increased plasma T3 was observed in some of these patients. The median
value of plasma rT3 in these patients is also higher than in normal subjects
 HI
although the median plasma T4 is subnormal. This may again indicate a
preferential secretion of rT3 by the thyroid gland; a preferential secretion
of rT3 and T3 over T 4 in response to TSH stimulation has recently been
reported in the thyroid of the dog (Laurberg 1978). It has been suggested
that the overproduction of T } in hyperthyroidism is caused by a thyroid
stimulating factor (immunoglobulins?) present in plasma (Izumi and
Larsen 1977).
Obviously, an increase in plasma T 3 (and rT3) may also be caused by an
increased rate of peripheral T4 conversion. However, thyroid tissue
concentration of T3 is relatively more increased than of T4 in hyperthyroid
patients (Larsen 1975), and the increase of plasma T3 thus seems primarily
caused by increased T3 secretion by the thyroid gland. On the other hand, it
is clear that in hyperthyroidism more T4 is available for peripheral
conversion, and consequently extra-thyroidal production of T3 could be
increased as compared to normal subjects, but this does not necessarily
imply that the conversion rate of T4 into T3 is increased (Schimmel and
Utiger 1977). Production rates and metabolic clearance rates of both T3
and rT3 are increased in hyperthyroidism and decreased in hypothyroidism
in man (Smallridge et al 1978). These authors have also estimated the
relative production rates (PR) of rT3 and T4, and reported a trend to higher
rT 3 /T 4 PR ratio's in hyperthyroidism and to lower rT 3 /T 4 PR ratio's in
hypothyroidism. Inada et al (1975) have found that the rate of
extrathyroidal conversion of T4 to T3 averages 3.00 ± 0.68% of the
extrathyroidal T4 pool per day in normal subjects, but is increased to 4.16
± 0.72% in untreated hypothyroid patients; thus, although the absolute
extrathyroidal production of T3 in normal subjects is greater than in
hypothyroid patients, in hypothyroidism relatively more T3 is derived
from the extrathyroidal deiodination of T4. This is in agreement with the
study of Maguire et al (1976) who report an increased conversion rate of T4
to T3 in hypothyroidism, and also in hyperthyroidism, with a fall to normal
after treatment. Therefore, it can be concluded that plasma concentrations
of T3 and rT3 are modulated in hyper- and hypothyroidism by both
thyroidal and extrathyroidal factors, as they are in the normal subjects.
In our patients the plasma ratio's of T 3 /T 4 are much more increased in
hypothyroidism than in hyperthyroidistn; since T3 and T4 change in the
same direction in these states, this indicates a relative overproduction of T3
to T4, which is greater in hypothyroidism than in hyperthyroidism. In
contrast, the plasma ratio's of rT 3 /T 4 are very much increased in
hyperthyroidism and about normal in hypothyroidism; since in these
states rT3 and T4 also change in the same direction, this may be interpreted
as indicating a relative overproduction of rT3 in hyperthyroidism as
compared to hypothyroidism.
Finally, we observed a steady decline in the plasma ratio's of rT3/T3
from severe hyperthyroidism to severe hypothyroidism, indicating that in
 112 a
l
hyperthyroidism a relative overproduction of rT3 occurs, while in ; ^= j
hypothyroidism there is a relative overproduction of T3. One can therefore , |?,
speculate that in hyperthyroidism more rT3 and less T3 is generated from fg!
T4, and that in hypothyroidism less rT3 and more T3 is generated from T4. ||7
The homeostatic significance of such a mechanism is clear: by modulating i * fcc.
e
the conversion of T4 into either active T3 or the metabolically inactive rT3 If
the peripheral tissues may counteract to some extent the deleterious effects ' ij
c
of excessive or insufficient thyroid hormone secretion. We are well aware
0
that our patient data do not allow any firm conclusions on the extent or the
side of the overproduction of the various iodothyronines in different states
of thyroid function. Clearly more studies are needed.

4b. Hypothyroidism. . =
|
Plasma T3 was normal in 18% of the hypothyroid patients of the groups •
E and F, and this limits the value of plasma T3 determinations foi the , :
diagnosis of hypothyroidism. Earlier studies of Mitsuma et al (1971) and j
Surks et al (1972) reported decreased plasma T3 values in all hypothyroid
patients (probably because only severely hypothyroid patients were
studied), but in all later studies normal plasma T3 values are found in a
portion of the hypothyroid patients: in 25% by Patel and Burger (1973), in
26% by Chopra (1974), in 12% by Burman et al (1975), in 43% by Seth et al
(1976), and in 48% by Stepanas et al (1977). These studies include 217
patients, and 83 of them (38%) had a normal plasma T3 concentration. Our
incidence is lower (18%), and this may be caused by the warning limits that
we apply for the normal range. It is obvious that plasma T 4 (or the FT4
index) is a more sensitive indicator for hypothyroidism than plasma T3,
although both tests are in no way specific for the diagnosis
hypothyroidism: decreased values can be caused by impairment of T4-»T3
conversion and abnormalities in the protein binding of thyroid hormones.
The basal TSH value and the TSH response to TRH appear to be better
indicators for the hypothyroid state, since 6 patients were found with nor-
mal T3 and T4 values, but increased TSH responses to TRH; three of these
patients had normal basal TSH values. Such patiens have been labeled as •];.|
having subclinical or preclinical hypothyroidism (Evered and Hall 1972; \
Evered et al 1973), a term used to define an asymptomatic state in which a f
i reduction in thyroid activity has been compensated for by an increased TSH !]
i output to maintain a euthyroid state. Neither term is entirely satisfactory, U
j because some patients of this group with minor complaints may improve by I
! L-thy roxine therapy and because the natural history of the thyroid function ||
disorder in this group of patients is not necessarily progressive: in some plas- \
< ma TSH reverts into the normal range, in some plasma TSH remains slightly .j
- elevated and in others a further increase in plasma TSH is seen together with j
{ the development of clinical hypothyroidism (Tunbridge et al 1978). Others •'
I have used the term preclinical hypothyroidism to indicate states with circu-
 113
lating thyroid antibodies (Bastenie et al 1971) or with hypercholesterol-
V'<' t
aemia (Fowler et al 1970) claiming an association with ischemic heart dis-
ease. However, subsequent studies have not always supported the concept
of hypercholesterolaemia as a premonitory sign of hypothyroidism (Nilsson
et al 1976; Tunbridge et al 1977; Gordin et al 1979), and also mean TSH
levels in subjects with antibodies to thyroid tissue are higher than in
controls in some studies (Bastenie 1977; Gordin et al 1979), but not in
others (Tunbridge et al 1977). Wilkin et al (1977) and Sluiter (1979) have
presented a kinetic model which explains the increased release of TSH in
subclinical hypothyroidism by a reduced functioning thyroid mass. Tests
of peripheral tissue function may be normal in subclinical hypothyroidism,
as demonstrated by a normal Achilles tendon reflex time in clinically
euthyroid subjects with high TSH levels living in an area of severe endemic
goitre (Goslings et al 1977). Thus, it may well be that the sensitivity for
thyroid hormone of different tissues can vary. However this may be, there
are subjects judged clinically to be euthyroid and with normal plasma
concentrations of T3, T4 and TSH, who have an increased TSH response to
TRH (Alaghband-Zadeh et al 1977); these subjects may be at risk for
developing hypothyroidism.
It can be concluded that the TSH response to TRH is the most sensitive
indicator presently available for detecting early hypothyroidism, and
further that hypothyroidism is a graded phenomenon. The pattern of
thyroid function tests in our patients of group D to F closely resembles the
pattern described by Evered et al (1973), Wenzel et al (1974), Bigos et al
(1978) and Kahn (1978). Detection and classification of hypothyroidism
by means of laboratory tests has proven to be superior to clinical
examination, but it remains to be proven if patients with subclinical
hypothyroidism benefit from treatment.
In earlier studies on the TSH response to TRH it is often stated that
hypothyroid patients have an exaggerated TSH response (Ormston et al
1971), and indeed the absolute increment of TSH progressively increases
when hypothyroidism is becoming more severe (table IV. S) However, the
relative increment of TSH progressively decreases at the same time (figure
IV. 2). The same trend is found in other studies (Evered et al 1973; Wenzel
et al 1974; Bigos et al 1978). This indicates a progressive decrease in the
release of TSH by TRH with the advancement of more severe
hypothyroidism. In this respect it is of interest that in the first weeks after
instituting replacement therapy in hypothyroid patients an enhanced TSH
response to TRH relative to the pre-treatment response is observed (Fischer
et al 1978; Ridgway et al 1979). Both phenomena may be explained by a de-
crease of the TSH content in the pituitary thyrotrophic cells, which is re-
stored during substitution therapy. Indeed, a low content of stored hormone I
is suggested by the paucity of secretory granules observed inTSH-produc-
ing pituitary adenomas secondary to primary hypothyroidism (Samaan et
 114
4v
al 1977). Sixteen of our 27 hypothyroid patients had the peak TSH value at
60 minutes after TRH (in contrast to five of the 63 normal subjects), and this
delay may also indicate a decrease in the directly releasable pool of stored i
TSH in the pituitary thyrotrophs. •O7

The T3 response to TRH in hypothyroidism must be analysed by the

absolute increment of T3, because the relative increments are not reliable
due to the low plasma concentrations of T3. Although the T3 response to
TRH is impaired in all our patients with overt hypothyroidism except one
(group F) which is in accordance with other studies (Shenkman et al 1972;
Squire and Gimlette 1977; Bigos 1978), measuring the T 3 response to TRH
has little to offer in the diagnosis of hypothyroidism. In fact, the only
indication for TRH stimulation in the process of diagnosing
hypothyroidism, is a normal or borderline elevated plasma TSH value
when T 3 and T 4 are also normal.

4c. Hyperthyroidism.
The preponderance of females in the hyperthyroid patients of group A
and B (with a female/ male ratio of about 4:1) and the older age of patients
with toxic nodular goitre as compared to patients with thyrotoxic Graves'
disease, is in agreement with the literature (Werner 1978). In general
thyrotoxic Graves' disease is associated with higher plasma T 3
concentrations than toxic nodular goitre, and this has also been described
by others (Patel and Burger 1973; Zurcher et al 1973; Shalet et al 1975;
Marsden and McKerron 1975; Sobrinho etal 1977). Plasma T3 is increased \\
in all patients with unequivocal hyperthyroidism in contrast to plasma T4;
thus T3 is a more sensitive test for detecting hyperthyroidism than T4, but
again the specificity of the T3 assay in this respect is decreased by the occur-
rence of increased T3 values due to other causes (e.g. increased binding to
plasma proteins, preferential secretion of T3 in subclinical hypothyroidism).

The incidence of 17% of T3-toxicosis in our series is greater than

- reported initially in the literature: 5% by Mitsuma et al (1971), 4% by
Hollander et al (1972), 11% by Bellabarba (1972), all in the U.S.A.; 8% by
Patel and Burger (1973) in Australia, 9% by Zurcher et al (1973) in
Switzerland, and 12.5% by Hollander et al (1972) in an area with endemic
goitre (Chile). Subsequently an ever higher incidence was reported, also
from areas without iodine deficiency: 8.2% by Turner et al (1975) in
Ireland, 10% by Carter et al (1975) in Australia, 17% by Marsden and
McKerron (1975) in England, 30% by Shalet et al (1975) in England, 14%
at first-onset thyrotoxicosis and 25% if recurrent thyrotoxicosis is included
by Stepanas et al (1977), also in the U.K., and lastly 31% by Sobrinho etal
(1977) in an area with endemicgoitre (Portugal). One may conclude from all
these studies that the real incidence of T3-toxicosis is unknown, but also that
the incidence even in areas without iodine deficiency is considerable in the
order of about 20% of all hyperthyroid patients. The incidence of T3 toxico-
 115
sis in the above-mentioned studies (as far as this could be ascertained), is -i - V'

11% if patients with recurrent thyrotoxicosis are excluded and 21% if these , !..
patients are included (the corresponding figures in our study are 13% resp. ] |:
17%). Thus, T3-toxicosis occurs relatively often in recurrent thyrotoxicosis. S . j|
This could be due to the development ofintrathyroidal iodine deficiency as a 1 •; ' %
result of the thyrostatic treatment and by the likelihood that recurrent > I-
thyrotoxicosis will be diagnosed earlier than first-onset hyperthyroidism. | |
T3 toxicosis occurs both in thyrotoxic Graves' disease and in toxic nodular •<
:
goitre. In the studies mentioned previously the nature of the hyperthyroid
state is given in 133 patients. 55% had Graves' disease, and 43% toxic nodu-
lar goitre. The corresponding figures in our study are 50% and 50% (exclud-
ing the patients with ophthalmic Graves' disease), as opposed to 60% resp.
38% in the patients of group A. T3 toxicosis thus occurs more often in toxic
nodular goitre than in thyrotoxic Graves' disease.
In theory, T 3 toxicosis might be caused by an enhanced peripheral
conversion of T4 into T3 Herrmann et al (1975) described two patients in j
whom excessive peripheral conversion of T 4 to T3 supposedly contributed t
to the development of T3 toxicosis. However, these patients were studied '
during antithyroid drug treatment. Also the observed ratio's of plasma rT3,
T 3 and T4 in our study are not in favour of such a mechanism. In this i
respect our results are in disagreement with other reports demonstrating |
normal or subnormal plasma rT3 values (Griffiths et al 1976) and j
decreased rT3/ rT3 ratio's (Wenzel and Meinhold 1975; Ratcliffe et al 1976) •
in patients with T 3 toxicosis as compared to normal subjects. Finally f]
Snarski and Snarska (1979) by measuring the daily degradation rate and the j
metabolic clearance rate of T4 found no indication for an increased
peripheral conversion of T4 to T3 in two patients with T3 toxicosis. It must !
be concluded that so far no patient has been described in whom T3
toxicosis is caused by an enhanced peripheral conversion of T4 to T3.

All patients of the groups A and B demonstrate a severely impaired or \

absent TSH response TRH. It is of interest that not infrequently patients \
are found with normal plasma T3 and T 4 values but an absent TSH ,;'
response to TRH (group C). This has been a common finding by many |
authors, especially in patients with ophthalmic Graves' disease, 8
hyperfunctioning thyroid adenoma or multinodular goitre, who are >;
judged clinically to be euthyroid (Lawton et al 1971; Ridgway et al 1973; 1
Chopra etal 1973; Franco etal 1973; Ormstonetal 1973; Clifton-Blighet |
al 1974; Gemsenjager et al 1976; Dige-Petersen and Hummer 1977; Elte et |
al 1977; Smeulers et al 1977; Kirkegaard et al 1977; Morgans et al 1978; |
Blichert-Toft etal 1978; Emrich and Bahre 1978). A possible explanation |
for this phenomenon is, that thyroid hormone secretion in these patients is ft
unduly high and sufficiently elevated (due to an expanded functioning '$,
thyroid mass) to inhibit the release of TSH by the negative feedback of 3
 116
»V-
thyroid hormones on the pituitary thyrotrophs. This situation can be 1
:'
considered as subclinical hyperthyroidism. The recurrence of a normal
TSH response to TRH after partial thyroidectomy in such patients, which
in some studies is accompanied by a significant decrease in plasma T4 and
T3 to within the normal range, also favours this view (Gemsenjager et al
1976; Morgans etal 1978; Blichert-Toft et al 1978). The suppression of TSH
release in these patients is not absolute, since oral administration of high
doses of TRH (40 mg) may result in TSH release in some of them (Staub et
al 1979; Kirkegaard et al 1979). This makes an abnormality in the process
of TSH synthesis or release in these patients less likely. Results of
peripheral tissue function tests (systolic time interval, sex-hormone
binding globulin and Achilles tendon reflex time) in patients with
subclinical hyperthyroidism are reported to be within the euthyroid range
(BirkhSuser et al 1979).
The T3 response to TRH as measured by the relative increment of T3 is
below normal in all hyperthyroid patients (groups A and B), and this is in
agreement with the findings of Murie et al (197S) and Squire and Gimlette
(1977). Clearly the expression of the T3 response to TRH by the relative
increment of T3 is superior to the absolute increment in hyperthyroidism
due to the wide fluctuations and more imprecise measurements of plasma
T3 concentrations in the very high range. As can be seen in figure IV. 3.,
there are two patients in group C with a normal increment of T3 (both
absolute and relatively) despite an absent TSH response. This suggests that
biologically active TSH is released in response to TRH, which is not
detected by the TSH radioimmunoassay (alternatively, it is possible that
TRH has a direct influence on the thyroid). Indeed, in a single case a TRH-
induced rise in TSH as assessed by an increased thyroidal iodine secretion
has been demonstrated when no substantial increase in immunoassayable
TSH could be detected (Boehm et al 1976). If so, the T3 response to TRH in
our hands is even more sensitive for measuring the negative feedback of
thyroid hormones at the pituitary level than the TSH response. This is
further illustrated by the following case history.

Patient X, a 69-years old female, was admitted to another hospital because of

depression and bizarre behaviour. Extensive investigation did not reveal any organic
cerebral disease or endocrine abnormality, with the exception of an absent TSH
response to TRH. She was referred to our clinic. Her behaviour had improved I
spontaneously, and a tentative diagnosis of hysteria was made. On clinical
examination she was euthyroid, and the FT4 index and the TSH- and T3-response to
i
TRH were all normal (figure IV. 4). Nine months later the patient returned from a
journey to the U.S.A. with the same bizarre behaviour. She was still clinically
euthyroid and the FT4 index was again normal; a hysterical twilight state was
diagnosed. The TSH-response to TRH was now absent, but the T, response was
normal.

Thus, an absent TSH-response to TRH can be caused by other states than

 " • * :

117
hyperthyroidism, especially by psychiatric disorders with a return to a
normal TSH-response after recovery (Loosen et al 1977; Gregoire et al
1977). Nevertheless, the maintained T3 response in our case indicates a
normal set-point of the pituitary-thyroid axis, and therefore euthyroidism.

. * - . :

The patients of group G, tentatively labeled as uT4-toxicosis" are

peculiar in that they do not fit in the described categories. These patients
are also peculiar from a clinical point of view: they were certainly not cases
of overt hyperthyroidism, and all five patients either suffered from a co-
existent non-thyroidal disease, or were or had been treated with drugs
known to influence thyroid hormone metabolism (propranolol, and
especially radiographic contrast agents). All patients had an abnormal
TSH and T3 response to TRH, compatible with hyperthyroidism. In
retrospect, hyperthyroidism at the time of testing seems probable in
patients 3 and S, possible in patients 1 and 4, and unlikely in patient 2.
Considerable debate has arisen in the literature if T4 toxicosis really
exists as a separate entity. Some consider the finding of raised FT4
index values together with normal T3 and FT3 index values as a normal
albeit unusual finding in elderly especially female subjects (Britton et al
1975), or merely as the borderline area of the upper normal range and the
lower hyperthyroid range (Hadden et al 1975), while others suggest that T4
toxicosis really exists (Turner et al 1975; Kirkegaard et al 1975; Turner et
al 1975; Engler et al 1978; Dorfman and Young 1978). The patients
described by these authors all have more or less severe co-existent non-
thyroidal disease, and very frequently have been exposed to iodine-
containing radiographic contrast agents; the only exception to this is the
one patient described by Dorfman and Young. More light upon this
problem has been shed by Birkhauser et al (1977). They describe 31
patients with various non-thyroidal diseases and a tentative laboratory
diagnosis of T4 toxicosis; none of the patients was clinically hyperthyroid
After recovering from their disease classic hyperthyroidism with a rise of
plasma T3 into the hyperthyroid range developed in 14 patients, a rapid
return of T4 values to normal was observed in 11 patients and in 6 patients
T4 remained elevated (in two of these transient iodine-induced hyperthy-
roidism was diagnosed with spontaneous return of T4 to normal as the
iodine was eliminated). Exposure to excess iodine was eventually traced in
I 16 of the 31 patients. In this respect a study of Sobrinho et al (1977) from
Portugal is of interest. These authors describe the preferential secretion of
T3 in 59 hyperthyroid subjects with Graves' disease or with nodular goitre;
it
I'
however in 5 out of 15 patients with iodine-induced thyrotoxicosis no
preferential secretion of T3 was noted, i.e. these patients had T4 toxicosis.
Most cases of T4 toxicosis therefore are due to impairment of the
extrathyroidal conversion of T4 into T3 caused by intercurrent non-
 118
thyroidal disease or by a particular medication (as shown by Biirgi et al
-a?
(1976) and Wu et al (1978) some iodine-containing radiographic contrast
agents inhibit the T3 generation from T4, but iodine itself has no effect on
I
the conversion of T4 as demonstrated by Burger et al in 1976). There are
only a few cases of T4 toxicosis in which T4 secretion by the thyroid gland is
really excessive in relation to T3 secretion, usually associated with
exposure to excess iodine. The transient increase in T4 observed in some
patients remains unexplained; it may well be caused by a decreased MCR
of T4 and/ or by abnormalities in the affinity or capacity of thyroxine
binding to the plasma proteins due to the intercurrent disease (recently
f
i'i'
Hennemann et al (1979) described elevated T4 and FT4 index but normal
FT4 values in euthyroid subjects, caused by an inherited increased affinity
of iodothyronines to the plasma proteins).
The incidence of "T4 toxicosis" in our series (groups A,B and G) is 7%, of
T3-toxicosis 15.5%, and of "classical" thyrotoxicosis 77.5% (two other
patients with T4 toxicosis are described in the chapters V and VII). Caplan
et al (1978) found T4 toxicosis in 12% of 83 elderly hyperthyroid patients.
This limits the value of the plasma T3 determination for the diagnosis of
hyperthyroidism. It is our policy to start with the assay of T4 and T3U, and
to proceed with a T3 determination if the FT4 index is normal and
hyperthyroidism is suspected; in all borderline cases a TRH stimulation
test is performed. A normal TSH response to TRH excludes the existence
of primary hyperthyroidism - this is the most important diagnostic aspect
of the TRH test. It should be realized however, that an absent or impaired
TSH response to TRH is not specific for hyperthyroidism, since blunted
responses are occasionally seen in normal subjects and psychiatric
patients.

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1 ' I

i ! • •''
f. & ' •
IK

I
?;<••

CHAPTER V. %
it
%-:
STUDIES IN PATIENTS WITH ACUTE NON-THYROIDAL II-
DISEASES
§:
V.I. Introduction. [< 7

In a comatose patient, a case of self-intoxication with dextromoramide

(Palfium), plasma T3 was found to be below the detection limit of the
assay, but plasma T4 and TSH values were normal. After recovery the
plasma T3 level returned into the normal range. In another patient
suffering from acute leukemia with a rapid downhill course, plasma T3
decreased progressively; it was speculated that the time of death would
coincide with the time at which plasma T3 would be undetectable. The date
of death was estimated as april 12th, the patient died on april 1 lth; plasma
T3 was undetectable in a blood sample drawn on april 10th (figure V.I).
These observations raised the following questions:
a. What is the incidence and what is the cause of a low plasma T3 concen-
tration in patients with acute non-thyroidal diseases?
b. Is there any relation between the plasma T3 concentration and the
severity of the disease?
c. Is a low plasma T3 level in these patients of any relevance in the set-
point of the pituitary-thyroid axis?
In an attempt to answer these questions a prospective clinical study was set
up.

V.2. Patients and methods.

Patients with an acute non-thyroidal illness in whom a complete

recovery was expected within two weeks, were studied. From the 28 initial
patients eight were excluded from the study: 4 patients could not be
observed for 2 weeks, and 4 were treated during the study period with
drugs that may interfere with thyroid function tests (radiographic contrast
agents, systemic heparin, diphenlhydantoin, heroin and methadone). The
remaining 20 patients were evaluated clinically after two weeks, and |
divided into four groups; group A (n=13) - full recovery, group B (n=
4) — improvement, but no full recovery, group C (n = 1) — no change in
the clinical condition, group D (n = 2) — deceased (see table V.I.).
 T.:ST*

125

5 10
Marctl

Figure V.I. Plasma T3 concentrations (• +) in a 63-years old male with acute leukemia,
who died at april 1 lth; the estimated time of death (by extrapolation of the two first plasma
T3 values, ( • — - • ) was april 12th.

Table V-l
Sex, age and diagnosis of 20 patients with an acute non-thyroidal disease.

group nr. sex age(yis) diagnosis

- full recovery in 2 weeks (n = 13)

1 F 15 infectious mononucleosis
2 M 15 meningitis
3 F 16 ihinophaiyngitis, meningism
4 M 21 chickenpox
5 M 33 toxicodermia
6 M 39 fever, polyarthralgia and rash of unknown
origin
7 M 40 self-intoxication
8 M 45 psittacosis
9 F 53 pyelonephritis
10 M 58 tonsillitis, pharyngitis
11 M 62 biliary colics
12 F 65 pneumonia
13 M 67 pneumonia

- improvement, but no full recovery in 2 weeks (n = 4)

14 M 27 meningitis
15 M 30 amebic abcess of liver
16 F 35 meningitis
17 M 38 active pulmonary tuberculosis

— no change in 2 weeks (n = 1) 1
18 F 47 sarcoidosis

- deceased within 2 weeks ( n = 2)

19 M 81 acute myocardial infarction
20 M 72 progressive obstructive jaundice due to
carcinoma of pancreas
 Table V-2
Thyroid function tests in 16 patients admitted because of acute non-thyroidal disease: group A (n = 12) - full clinical recovery in 2 weeks, group B (n = 4) -
clinical improvement but no full recovery in 2 weeks. (Values as mean ± SD, except plasma TSH and iodothyronine ratio's which are presented as the geometric
mean ± SD).
group day 1 2 3 4 5 6
i TSH mU/1 A 0.9 (0.4 - 2.1 ) 0.9 (0.3 - 2.8 ) 1.4 (0.6 - 3.1 ) 1.5 (0.9 - 2.4 ) 1.2 (0.7 - 2.1 ) 1.2 (0.7 - 2.0)
B 2.6 (1.5 7- 4.5 ) 1.3 (0.6 - 2.8 ) 1.2 (0.5 - 3.1 ) 1.1 (0.5 - 2.8 ) 1.2 (0.5 - 3.2 ) 1.1.(0.3 - 4.1 )
ft
} rTjiimol/1 A 0.42 ± 0.18 0.43 ± 0.17 0.42 ± 0.20 0.39 ± 0.17 0.34 ±0.12 0.33 ± 0.07
B 0.56 ± 0.20 0.54 ± 0.21 0.54 ± 0.30 0.58 ± 0.36 0.54 ±0.31 0.57 ± 0.33
Ts nmol/1 A 1.01 ± 0.33 1.06 ± 0.38 1.31 ± 0.54 1.48 ± 0.43 1.44 ± 0.37 1.42 ± 0.42
B 0.85 ± 0.46 0.94 ± 0.66 0.84 ± 0.48 1.02 ±0.54 0.91 ± 0.34 0.78 ± 0.41 4
T4nmol/1 A 87 ±22 86 ±22 981 18 106 ±19 102 ± 16 105 ± 20
B 85 ±28 96 ±39 88 ±26 96 ±36 94 ±26 90 ±22
TSU A 0.97 ±0.13 0.94 ±0.12 0.92 ±0.12 0.93 ±0.11 0.92 ± 0.10 0.93 ±0.11
B 1.04 ±0.19 1.02 ± 0.16 0.99 ±0.12 0.98 ±0.13 1.00 ±0.12 0.99 ± 0.12
FT4 index A 84 ±24 80 t 18 90 ± 17 99 ±21 94 ±17 99 ±26
B 85 ±23 96 ±41 86 ±26 94 ±42 93 ±28 88 ±25
rT,/T a xl0-' A 412(225 - 753 ) 403(208 - 781 ) 330(146 -•745 ) 261(150 - 452 ) 233(137 - 396 ) 236(147 - 378 )
B 788(326 - 1903 ) 731(247 - 2168) 696(251 - 1931) 540(204 - 1426) 564(250 - 1271) 742(276 - 1997)
rT,/T 4 xl0- J A 4.61(3.01 - 7.05 4.76( 3.15 - 7.18) 4.01( 2.61 - 6.15) 3.53( 2.50- 4.99) 3.22( 2.27 - 4.58) 3.11( 2.41- 4.02)
B 6.24(3.73 - 10.45 5.62( 3 . 3 0 - 9.57) 5.59( 3.27 - 9.53) 5.40( 2.92 - 9.97) 5.30( 2.96 - 9.50) 5.70( 3.69 - 8.81)
T s /T 4 xl0-' A 11.2 (7.9 - 15.8 ,> 11.8 ( 8.4 - 16.6 ) 12.2 ( 7.5 - 19.7 ) 13.6 ( 9.7 - 19.0 ) 13.8 (10.4 - 18.4 ) 13.2 ( 9.9 - 17.5 )
B 8.4 (5.0 - 14.0 ' 7.7 ( 4.1 - 14.4 ) 8.0 ( 4.5 - 14,2 ) 10.0 ( 7.0 - 14.3 ) 9.4 ( 7.4 - 11.9 ) 7.7 ( 4.2 - 14.1 )

g ? ^ ^ ^
 7 10 14 ±20 ±30 ±40 normal values

1.3 (0.8 - 2.0) 1.2 (0.8 - 2.0) 1.3 (0.8 - 2.1)

1.8 (1.0 - 3.2) 2.0 (1.0 - 3.9) 1.9 (0.7 - 5.6) 2.1 (1.0 - 4.6) 1.3 (0.3 - 6.0) 1.5 (0.5 - 4.8) 0.7 (<0.3 - 2.3)
0.29 ± 0.08 0.27 ± 0.07 0.27 ± 0.10
0.51 ± 0.30 0.50 ± 0.33 0.46 ± 0.23 0.30 ± 0.15 0.30 ± 0.11 0.34 ± 0.18 0.24 ± 0.07
1.50 ±0.45 1.56*0.47 1.63 ± 0.39
0.91 ± 0.37 0.84 ± 0.39 1.15 ± 0.45 1.21 ±0.27 1.95 ± 0.76 1.96 ±0.52 1.85 ± 0.37
104 ± 22 103 ± 20 106 ± 17
98 ±34 85*20 104 ±29 108 ± 32 106 ± 31 110 ±33 102 ±18
0.95 ± 0.10 0.92 ± 0.13 0.94 ±0.12
0.99 ±0.13 1.02 ± 0.15 0.99 ± 0.14 0.9S ± 0.10 0.95 ± 0.16 0.95 ± 0.14 0.94 ± 0.09
99 ±25 94 ±23 99 ±20
97 ±37 87 ±28 102 ± 27 104 ±39 101 ± 33 106 ±46 96 ±15
196(113 - 340) 172(104 - 285) 157( 95 - 258)
529(242 - 1155) 571(223 -1464) 391(193 - 793) 2*3(110 - 453) 157( 77 - 317) 162( 92 - 286) 124( 86 - 179)
2.74(2.00- 3.75) i3
2.54(1.78 - 3.61) 2.38(1.70 - 3.33)
4.82(3.39 - 6.84) 5.29(2.95 - 9.50) 4.15(2.68 - 6.42) 2.55(1.94 - 3.34) 2.79(2.08 - 3.73) 2.93(2.33 - 3.69) 2.23(1.62 - 3.08)
14.0 (10.1 - 19.3) 14.8 (11.0 - 1 9 . 8 ) 15.1( 11.8 - 19.5)
9.1 ( 5.9 - 14.2) 9.3 ( 6.3 - 13.7) 10.6( 8.0 - 14.1) 11.4 (7.1 - 18.4) 17.8 (11.1 - 28.4) 18.1 (12.1 - 27.1) 18.0 (14.9 - 21.9)
 128
Blood samples were taken on days 1,2,3,4,5,6,7,10 and 14; day 1 was the
day of admission. In the patients of group B several blood samples were also
$
taken after two weeks. In each plasma sample the following
determinations were done: TSH, rT3, T3, T4 and T3U. The competitive
I
protein binding method of Murphy and Pattee was used for the assay of T4.
Stimulation tests with 200 jug TRH i.v. were performed as described in
chapter III.2 on days 1 and 14, if feasible. All samples of one patient were
assayed in the same run in order to avoid inter-assay variation.
Statistical evaluation of the trend in hormone levels and of the TSH- and
T3-responses to TRH stimulation was performed by analysis of variance
I:
(Bennett and Franklin 19S4). Because of their skewed distribution, plasma
TSH levels and the ratio's of plasma iodothyronine concentrations were
analyzed after logarithmic transformation. In the statistical analyses the
level of significance was taken as a = 0.0S; undetectable hormone
concentrations were taken as SO percent of the detection limit.

V.3. Results.

The sequence of plasma T4, T 3 and rT3 concentrations in patient nr 12 is

depicted in figure V.2. Plasma TSH was undetectable in this patient, and
the TSH- and T3-response to TRH were absent resp. subnormal both at
day 1 and at day 14. Subsequently hyperthyroidism was diagnosed, and
she responded favourably to antithyroid drug treatment. The data of this
patient were excluded from the further statistical evaluation.
On admission plasma T3 was subnormal (i.e. below the percentile P 2 . s of
the normal values) in 16 out of 19 patients (84%), and plasma rT3 was
supranormal (i.e. above the percentile P97.5) in 12/19 patients (63%);

14
days

Figure V.2. Plasma T4,T3 (solid lines) and rT3 (dashed line) concentrations in a 65-years old
female admitted because of pneumonia. The biochemical pattern of T4-toxicosis (day 1)
evolved to 'classical1 T r and T4-thyrotoxicosis at the time the intercurrent pneumonia had
subsided (days 10 and 14).
 129

I Table V-3
Thyroid function tests in two acutely ill patients, who died within two weeks after ad-
mission (group D).

patient D, patient D a
on admission - last sample on admission - last sample

IS TSH mU/1 1.3 0.5 0.4 < 0.3

rT3 nmol/1 0.68 1.10 1.44 3.15
in T3 nmol/1 1.11 0.59 0.86 0.68
T4 nmol/1 104 89 143 137
M
>1 T,U
FT« index
0.91
95
0.96
85
0.99
142
1.26
173
rT 3 /T,xl0- s 613 1864 1674 4632
rT,/T 4 xl0- 3 6.54 12.36 10.07 22.99
T,/T 4 xl0-» 10.67 6.63 6.01 4.96
peak TSH mU/1 11.6 - 4.4 -
peak T3 nmol/1 1.68 — 1.42 —
i;

plasma ,T4 and the FT4 index were subnormal in 6/19 patients (32%).
The results of the thyroid function tests in the patients of groups A and B
are presented in table V.2. In the patients of group A plasma T 3 ( p < 0.001),
T 4 (p < 0.001) and the FT4 index (p < 0.01) increased, whereas rT3 (p
< 0.01) and T3U (p < O.OS) decreased; plasma TSH did not change (p >
0.1). In contrast, no change in these parameters was found in the first two

3.0 r

10 2.0

1.0

01-

-30 0 20 60 120 180 240 -30 0 20 60 120 180 240

min min

Figure V.3. The TSH- and T,-response to 200//g TRH i.v. in 16 patients with acute non-
thyroidal diseases on admission (solid lines) and two weeks later (interrupted lines) (values as
mean ± SD).

t'7
 130

The TSH- and Ta-response to 200 fig TRH i.v. in 15 patients with acute non-thyroidal
disease on admission (~ day 1) and appr. two weeks later (~ day 14) after full clinical
recovery (group A, n = 11) or when clinically improved but not fully recovered (group B,
n = 4).
ii-'
(TSH values are presented as the geometric mean ± SD, T 3 values as x ± SD with in
brackets the range of the relative increment). •:•'•

basal value peak value absolute relative

i
group day of L~;
'if''
•

TRH- increment increment

test
I
TSH mU/1 A 1 0.9(0.4 - 2.2) 5.5(3.1 - 10.1) 4.5(2.5- 8.1)
14 1.3(0.8 - 2.1) 6.2(4.3- 9.0) 4.8(3.2- 7.2)
B 1 2.0(0.7 - 5.7) 8.5(4.3 - 16.6) 6.3(3.4 - 11.6) I',
14 1.9(0.7 - 5.6) 10.7(5.6 - 20.7) 8.5(4.4 - 16.1)

T3nmol/1 A 1 1.02 ± 0.34 1.55 ± 0.58 0.53 ±0.28 5 2 ( 2 1 - 80)

14 1.59 ± 0.40 2.29 ± 0.65 0.71 ± 0.29 4 4 ( 2 0 - 62)
B 1 1.01 ± 0.66 1.68 ± 0.79 0.68 ± 0.32 111(29 - 247;
14 1.15 ± 0.45 1.97 ± 0.53 0.82 ±0.22 83(41 - 140)

weeks in group B. However, when the assay results of the subsequent

weeks were also analyzed, plasma T3 (p < 0.01), T 4 (p < 0.01) and the FT4
index ( p < 0.01) increased, whereas rT3 decreased ( p < 0.02); T3U and TSH
did not change. Also, the ratio's rT3/T3 and rT 3 /T 4 decreased (p < 0.01)
and T3/T4 increased ( p < 0.001) in group A, but did not change in the first
two weeks in group B.
The results of the thyroid function tests in the patient whose clinical
condition remained unchanged (group C), were almost the same at day 1
and 14: TSH 1.3 vs. 1.2 mU/1, rT3 0.28 vs 0.24 nmol/1, T3 1.29 vs. 1.23
nmol/1, T4 68 vs. 67 nmol/1, T3U 1.02 vs. 1.13, FT4 index 69 vs. 75. In the
s two patients who died (group D) a progressive decrease in plasma T 3 and
increase in plasma rT3 was observed (table V.3).
The TSH- and T3-response to TRH on admission and appr. two weeks
later of 16 patients (nrs. 1-5, 7-11, 13-18) are depicted in figure V.3. The
TSH- and T3-responses were normal on both occasions, and no difference
was observed between day 1 and day 14 (although T3 levels were higher on
day 14). Evaluation of the responses in group A and B separately also
failed to reveal a difference between day 1 and 14 (table V.4.). In the one
patient of group C the peak TSH values after TRH were 6.9 and 6.3 mU/1,
and the peakT3 values 1.60 and 1.63 nmol/1 on days 1 and 14 respectively.

I-
 ," -»'••'

131
D V.4. Discussion.

|f The high incidence of a subnormal plasmaTy (84%) and a supranormal

p rT3 level (63%) in our patients with non-thyroidal diseases is in agreement
ft with a reported incidence of 71,59, 80 and 77% for low T3 values and of ?
% 53% for high revalues (Bermudez etal 1975, Burrows etal 1975, Lim etal
I; 1977, and Chopra et al 1979). Thus it appears that subnormal T3 levels,
jfe often in combination with supranormal revalues, are a common finding ; g;
|1 in patients with acute or chronic non-thyrpidal disease (Carter etal 1974; , ;> ft
| Chopra et al 1974; Bermudez et al 1975; Burrows et al 1975; Chopra et al
p 1975, Green etal 1975; Hflfner and Gless 1975; Nomura etal 1975;Burgeret
5 al 1976; Gavin et al 1976; Jarnerot et al 1976; Ramirez et al 1976;
\ Brinckmeyer et al 1977; Burrows et al 1977; Finucane et al 1977; Lim et al
\ I977;Ljunggrenetall977;Rudmanetall977;Talwaretall977;Wartofsky :
f et al 1977; Kolendorf et al 1978; Maharajan et al 1978; Naeye et al 1978; \
\ Ratcliffe etal 1978; Saunders etal 1978; Afrasiabi etal 1979; Helenius and i j
Lieuwendahl 1979; Pittman et al 1979; Rose and Davis 1979; Weissel et al
1979).Thenatureofthediseaseseemstobeirrelevantforthedevelopmentof j
the 'low T3 syndrome', since subnormal T3 values have been observed in !
almost every disease so far studied. The nonspecific nature of the reciprocal
changes in plasma T3 and rT3 is further indicated by similar alterations in the
plasma iodothyronines after surgical stress (Burr et al 1975; Adami et al
1978; Chan etal 1978; Hagenfeldtetal 1979; Kehletetal 1979; Prescott etal :
1979). !

The reciprocal changes in plasma T3 and rT3 strongly suggest that they I
are caused by modulation of the peripheral conversion of TA. Kinetic ;
studies in patients with hepatic cirrhosis (Nomura et al 1975; Chopra 1976; \
Lumholtz et al 1978), renal failure (Lim et al 1977), or diabetes (Pittman et
al 1979) indicate impaired production rates (but normal metabolic ;
clearance rates) of T3 and normal or slightly increased production rates ) I
(but decreased metabolic clearance rates) ofrT3, whereas the production of | :
T4 is equal to or slightly less than that found in control subjects.The fall in ; |
[
plasma T3 and rise in plasma rT3 can thus be explained by impairment of i
the T4-»T3 and rT3*3,3'-T2 deiodinative process due to inhibition of 5'- j • |
deiodination. It has been speculated that the decrease in 5'-deiodinating \ f
activity is related to the stress-induced rise in catecholamines and • \
especially in cortisol secretion, since corticosteroid medication in man also :': [
results in low T3 and high rT3 plasma concentrations (Chopra et al 1975). A [
However, no relation has been found between plasma cortisol and thyroid ^ I
hormone levels after surgery (Kirby et al 1973; Prescott et al 1979), and | j
inhibition of the glucose and cortisol response to hysterectomy by %
blockade of afferent pathways with spinal anaesthesia (Madsen et al 1977) %
 132
fcl.
jV,

'IS 2.00

I 1.50

1.25 1.00

sr
•,1.00 L0.75

0.75 0.50

L
0.50 0.25

14 14
days days

Figure V.4. Mean plasma T3 and rT3 concentrations in 18 patients with acute non-thyroidal
diseases, who after admission (day 1) made a full (group A, n=12) or incomplete (group B,
n=4) clinical recovery in 2 weeks, or who died (group D, n=2).

did not prevent the postoperative decrease in T3 (Brandt et al 1976). As to

I
the catecholamines, it is difficult to reconcile a possible direct inhibition of
the T4-»T3 conversion by catecholamines with the similar inhibitory effect
of propranolol (cf. chapters VII and VIII).
Glucose metabolism in the hexose-monophosphate shunt is decreased
during illness, resulting in a lower concentration of NADPH, which is the
cofactor in the reduction of glutathione. Visser (1978) has put forward the
hypothesis that the modulation of the conversion of T4 during illness is
related to a lower intracellular reduced glutathione concentration, since
reduced glutathione probably serves as a cofactor for the S'-deiodinating
enzyme. Fasting per se results in similar changes, however, not all acute or
chronically ill patients reduce their dietary intake. The biological
significance of the low T3 syndrome in non-thyroidal disease is unknown,
but it may be speculated that the low T3 syndrome fits into the general
adaptation syndrome in which the body seeks to diminish the catabolic
effects of stress (see further general discussion, chapter IX).

In our patients, plasma T3 increased and rT3 decreased concomitantly

with the improvement in clinical condition; in fact, these changes occurred
also within the normal range, and were observed in every patient. The same
sequence of events has been observed by others in (sometimes experimen-
tally induced) non-thyroidal disease and after surgery (Burr et al 1975;
Burger et al 1976; Talwar et al 1977; Wartofsky et al 1977; Adami et al 1978;
Chan etal 1978; Naeyeetal 1978; Maharajanetal 1978; Saundersetal 1978;
Hagenfeldt et al 1979; Kehlet et al 1979; Prescott et al 1979). A relation
 133
v,'v. between the plasma T3 concentration and the prognosis in non-thyroidal
disease is strongly suggested by the rather slow return of T 3 and rT3 values
into the normal range if clinical recovery was delayed, and by the further
decrease in T3 and increase in rT3 in the patients who died (figure V.4). A pre-
terminal fall in plasma T3 is also reported by McLarty et al in 1975 and by
Maharajan et al in 1978. It has been our experience that patients are
especially at risk for an unfavourable course of their disease if plasma T3
decreases below 1 nmol/1 and plasma rT, increases above 1 nmol/1.
A relation between the severity of a disease and plasma T3 is also suggested
by other studies. The fall in plasma T3 is related to the degree of renal failure
(Ramirez et al 1976; Finucane et al 1977; Kolendorfet al 1978; Weissel et al
1979), to the degree of liver damage as estimated by the plasma albumin
concentration (Nomura et al 1975; Green et al 1977), and to the degree of
fever in patients with infectious diseases (Ljunggren et al 1977; Maharajan et
al 1978). Patients with malignant lymphoma stage IV B have lower plasma
T, concentrations than patients in stage I-IV A, and the prognosis of
patients with lung cancer is worse if they have a subnormal T3 (Ratcliffeetal
1978). A more quantitative approach relating plasma T3 and rT3 levels to the
severity of a disease, is offered in the next chapter.

The set-point of the pituitary-thyroid axis in the 'low T3 syndrome' is of

considerable interest. The low concentration of plasma T3 is usually
associated with a low free T3 concentration (Chopra et al 1974; Chopra et al
1975; Talwar et al 1977; Chan et al 1978; Kehlet et al 1979; Prescott et al
1979), and since even small decreases in plasma thyroid hormone
concentrations are followed by hyperresponsiveness of TSH to TRH
stimulation (Vagenakis et al 1974) one would expect an increase in basal and
stimulated TSH values in these patients. As our results indicate, this is not
so: despite substantial decreases in plasma T3 concentration the TSH levels
are not elevated, nor is the TS H- and T3-response different on day 1 and day
14, and in this respect the patients of group A and B behave similarly.
Apparently the pituitary thy rotrophic cells do not react to the low plasma T3
levels: the pituitary-thyroid axis functions as if a euthyroid state exists. A
normal increase in T3 levels in patients with non-thyroidal disease in
response to either exogenous TSH or a TRH-induced rise in endogenous
TSH, has been reported by others (Carter et al 1974; Chopra et al 1974;
Carter et al 1976; Talwar et al 1977; Weissel et al 1979), and also a normal
TSH-response to TRH is found by most authors. Therefore patients with
the low T3 syndrome are often called 'sick euthyroid patients', also because
they do not present with signs or symptoms of hypothyroidism. Occasion-
ally however slightly elevated basal TSH values and increased TSH
responses to TRH are reported, especially in liver disease (Chopra et al 1974;
Nomura et al 1975; Bermudez et al 1975; Green et al 1977; Rose and Davis
1979), whereas a blunted TSH release has also been observed, especially in
f:

St:
 134 ?
renal failure (Lim et al 1976; Ramirez et al 1976; Hooper 1976; Naeye et al •)
1978; Weissel et al 1979). It thus cannot be denied that in the low T 3
syndrome adaptations at the hypothalamic/ pituitary level may occur.
However, the magnitude of the adaptation of thyroid hormone metabolism ; p
tostressfullconditionsseemstobemuchgreaterintheperipheraltissues.lt : , l£
is noteworthy that patients dying of a chronic wasting illness show a striking j fe;
reduction of the concentration of T 3 in the heart, liver a n d kidney, together j ||
with a relative increase of T 4 in these tissues (Reichlin e t a l 1973). Thus, there E;
seems t o b e a discrepancy between some sort of'hypothyroidism'at the level j
of the peripheral tissues, a n d the 'euthyroidism' a t t h e central hypothalamic- '
pituitary-thyroid level. Ideologically, the low T 3 concentration could help
to mitigate the excessive catabolism in the peripheral tissues; in this
situation a stimulation of T S H release a n d thereby of thyroid h o r m o n e
secretion would serve n o useful purpose. It is unlikely that in t h e low T 3 , |
syndrome t h e absence of a n increase in T S H release is effectuated by a |
suppressive effect of the high r T 3 concentrations (Nicod etal 1976). T h e ro/e | ! {
!
ofthyroxine seems to be crucial in this respect.
i :
Plasma T4 was subnormal in one third of our acutely ill patients, which is
in agreement with reports of others (Carter et al 1974; Chopra et al 1974, ^
197S; Burger et al 1976; Adami et al 1978; Naeye et al 1978; Saunders et al |
1978; Ratcliffe et al 1978; Kehlet et al 1979; Chopra et al 1979). A major |
determinant of the fall in plasma T4 induced by illness or surgery, is a •',
decrease in binding capacity of the plasma proteins (especially of TBPA) for .;
thyroid hormones, resulting in an increase of the T3U values (Surks and :
Oppenheimer 1964; Bellabarba et al 1968; Lutz et al 1972; Kirby et al 1973;
Bermudez et al 1975; Talwar et al 1977; Adami et al 1978; Naeye et al 1978;
:
Chopra et al 1979; Helenius and Liewendahl 1979). It has been argued that
unknown substances circulate in the plasma of these patients that may ;
inhibit the binding of T4 to the plasma proteins (Chopra et al 1979; Helenius •
and Liewendahl 1979). This would explain why the FT4 concentrations (as :
measured directly by dialyses) is normal or even elevated in 'sick euthyroid \
patients' (Burrows et al 1977; Brinckmeyer et al 1977; Talwar et al 1977; ;
Chopra etal 1979; Kehlet etal 1979; Prescott et all 979). In this situation the h
FT4 index—as calculated from T4 and T3U — is often low and is, therefore, JJ \
not a reliable parameter. ' -
Moderately elevated T4 values have also been reported, especially in I
elderly patients (Burrows et al 1975,1977; Wartofsky et al 1977; Chan et al >]
1978; Maharajan et al 1978; Ratcliffe et al 1978; Prescott et al 1979). One 2
then finds a pattern of T 4 -toxicosis\ The reason why in some elderly qj
patients a n increase in T 4 is found is not clear, but may well be due t o a n j|

I impairment of the deiodination of T 4 .

The elegant studies of Silva and Larsen have recently clarified some
aspects of the feedback control of thyroid hormones at the pituitary level
c(

'\
i

I
 135 t

i.
- 50

X 40 S
£

to*

l
Figure V.5. Plasma T4, T3 and rT3 in a 74-years old female, who died 7 days after admission
because of progressive cardiac failure due to mitral insufficiency. Hypothyroidism was
recognised after death (TSH values in all samples greater than 100 mU/1). Note the absence
of a pre-terminal rise in rT3, whereas T3 continuously decreases.

(SilvaandLarsen 1977,1978;Silvaetal 1978,1978; Larsenetal 1979). They

demonstrated in the rat: a) that there is an excellent correlation between the
nuclear T3 content of the pituitary thyrotrophs and the release of TSH, b)
that the anterior pituitary converts T4 into T3, c) that pituitary nuclear T3 is
derived from intrapituitary T4 monodeiodination and also directly from
plasma T3, d) that local T4 monodeiodination contributes relatively more to
the nuclear T3 than plasma T3 in the pituitary, as compared with liver and
kidney, and e) that intrapituitary T4-*T3 conversion is blocked by iopanoic
acid but not by propylthiouracil, whereas both agents inhibit T4-iT3
conversion in liver and kidney. Thus the pituitary thyrotroph and the
peripheral tissues can respond differently with regard to the conversion of
T4, and it may well be that the capability of the pituitary thyrotroph to derive
its nuclear T3 from local T4 conversion to a much greater extent than the
peripheral tissues, answers the question of 'central euthyroidism' and
'peripheral hypothyroidism' in non-thyroidal illness. It underlines the
uncertainty with regard to the real metabolic situation at the cellular level,
which may not always be reflected correctly by the levels of the circulating
thyroid hormones; the tissues may locally regulate the production of T3
according to their need (Obregon et al 1979).

The non-specific changes in plasma iodothy ronine concentrations caused

by non-thyroidal disease compromise the usefulness of plasma assays in the
diagnosis of thyroid disease in a sick patient. If T4 and FT4 index values are
normal, thyroid disease is probably absent. If they are abnormally low, the
finding of a high rT3 concentration in plasma indicates euthyroidism rather "•i
than hypothyroidism (see fig. V.5). Finally, it can be concluded that even in
sick patients the TRH test maintains its great diagnostic value as witnessed
by the responses seen in all our acutely ill patients.
 136
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Afrasiabi M A, Vaziri N D, Gwinup G et al. Thyroid function studies in the nephrotic
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Bellabarba D, Inada M, Varsano-Aharon N, Sterling K. Thyroxine transport and turnover in
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ft
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I
41:27-40. .. J .
Brandt M R, Kehlet H, Skovsted L, Hansen J M. Rapid decrease in plasma tniodothyronine
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Brinckmeijer L M, Woem A M, Nissen N I. Thyroid function in malignant lymphoma. Acta
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Burger A, NicodP, SuterP, VallottonMB, VagenakisA, BravermanL. Reduced active
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hormones in the elderly sick: "T4 euthyroidism". Brit Med J 1975; 437-9.
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function. Lancet 1974; 2: 971-4.
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subnormal serum triiodothyronine. J Clin Endocrinol Metab 1974; 39: 510-11.
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 137
'; i
GreenJRB, SnitcherEJ, MowatNAG, EkinsRP, ReesLH, DawsonAM. Thyroid { ,i
•i.v. function and thyroid regulation in euthyroid men with chronic liver disease: evidence f.
of multiple abnormalities. Clin Endocrinol 1977; 7: 453-61. , |
%': Hagenfeldt I, Melander A, Thorell J, Tibblin S, Westgren U. Active and inactive thyroid hor- :• »
;f; mone levels in elective and acute surgery. Acta Chir Scand 1979; 145:77-82. .. %
'•: Helenius T, Liewendahl K. Abnormal thyroid function tests in severe non-thyroidal illness: j ,, |
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^- Hiifner M, Gless K H. Die euthyreote hypotriiodothyroninamie. Schweiz Med Wschr 1975;
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•fi

CHAPTER VI.

STUDIES IN PATIENTS WITH ACUTE MYOCARDIAL IN-

FARCTION ^ _ ^ ^

VI. 1. Introduction.

In patients with non-thyroidal disease a qualitative relationship exists

between the severity of the disease and the changes in the peripheral
conversion of T 4 (see chapter V). A quantitative relationship is more
difficult to establish, since the severity of a disease cannot be graded easily.
The entity of acute myocardial infarction (AMI) is exceptional in this
respect, because the infarct size (which can be estimated by serial
measurements of serum enzyme concentrations) is directly correlated to
the severity of the condition: a greater infarcted area is associated with
more complications and a worse prognosis (Chapman 1972).
We therefore studied prospectively thyroid hormone metabolism in
patients with an acute myocardial infarction, in order to detect a possible
quantitative relation between the infarct size and the changes in the
peripheral conversion of T4. The pituitary-thyroid axis was also evaluated
by serial measurements of plasma TSH, since conflicting reports have been
published on thyroid function in AMI: a normal, a decreased or an
increased secretion of T4 and of TSH have been suggested (Ceremuzynski
et al 1970; Saeed-Uz-Zafar et al1971; Harland et al 1972; Vanhaelst et al
1976; Kaplan et al 1977; Westgren et al 1977; Smith et al 1978; Ljunggren et
all979).

s
VI. 2. Patients and methods.

A series of consecutive patients admitted to the coronary care unit

because of a suspected AMI, was studied. Acute myocardial infarction was
diagnosed on a characteristic history correlated with typical electrocardio-
graphic changes and a serial rise in the serum enzymes glutamic oxalacetic
transaminase (SGOT), glutamic pyruvic transaminase (SGPT) and lactic
dehydrogenase (LDH). Infarct location was defined according to the
criteria proposed by the New York Heart Association (1973). The size of
infarction was estimated by 6-hourly measurements of SGOT until the
peak value was reached (West et al 1966; Killen and Tinsley 1966; Kibe and
Nilsson 1967). From the initial 43 patients seven were excluded from the
Data of 36 patients admitted because of acute myocardial infarction (AMI).
1
nr sex age HAT peak SGOT localization infarct complications
hrs U/l pump rhythm conduction
failure disturbances
1
uncomplicated AMI (group A, n = 17).
1 M 48 2.0 23 anterolateral
2
3
M
F
59
71
15.5
3.0
38
40
anterolateral
inferolatcral
i
1 A
4 M 67 3.75 45 anterolateral i
5 M 57 1.0 49 lateral
6 F 59 4.5 50 anteroseptoinferolateral ) ' !'
7 M 66 4.5 62 anteroseptal
8 M 54 4.0 68 anterolateral ;!
9 F 88 2.5 68 anteroseptolateral '"]
inferior *(
10 M 77 13.0 71
11 M 65 -2.0 74 anteroseptal
12 M 67 4.0 85 inferoposterolateral 'J
13 M 51 3.5 115 inferoposterolateral ;
14 M 58 5.0 116 inferolateral
15 M 59 14.5 125 anteroseptolateral • • ' • ' • • ; ' '

16 M 72 5.0 139 anteroseptolateral '" ', ' :

17 M 86 31.0 168 anterolateral • ' ' !

x(range) 65(48 - 88) 6.8(-2 - 3 1 ) 79(23 - 168) i , * ,' ''

AMI ferouD B, n = 17)

complicated AMI (group B, n = 17)
18 M 66 24.0 34 infcrolateral X
19 M 60 1.25 39 anteroiateral AF
20 F 76 3.0 61 anterolateral AF
21 F 62 2.5 81 inferolateral X AF CB
22 M 67 0.25 105 anterolatcral AF
23 M 60 1.0 119 inferoposterior X CB
24 M 65 14.5 122 infcropostcrolatcral X
25 M 78 5.5 143 anteroseptal X
26 M 72 1.0 149 inferoposterolateral X AF SA
27 M 45 18.5 150 anterolateral X
28 M 66 4.0 168 inferior X AV
29 M 51 4.0 172 anterolateral AF
30 M 45 4.5 196 inferoposterior AV
31 M 57 1.0 200 inferoposterolatcral X AF
32 M 54 1.75 225 inferior VF CB
33 M 49 2.0 251 anteroscptal VT
34 M 47 2.75 257 antcroseptolateral X VF
x(range) 60(45 - 78) 5.4 (0.25 - 24) 145(34 - 257)

fatal AMI (group C, n = 2)

35 F 72 2.5 64 anterolateral rupture of papillary muscle
36 M 66 4.0 167 inferoposterior cardiac tamponade

all patients (n = 36)

30M
x(range) 6F 63(45 - 88) 5.9 (-2 - 31) 112(23- 257) 9 antero/septal
12 antero/scptal
15 infero/posterior

Abbreviations: SGOT = serum glutamic oxalacetic transaminase; HAT = hospital arrival time; AF = atrial fibrillation; VF = ventricular fibrilla-
tion; VT = ventricular tachycardia; CB = complete (third degree) AV block; SA = sino-atrial conduction disturbances; AV = atrioventricular con-
duction disturbances other than CB.
 142 V:
study: 3 patients had no AMI, and 4 were treated during the study period ; %
with drugs that interfere with thyroid function tests (B-adrenoceptor |<3
blocking agents, oral contraceptives, anabolic steroids). Thyroid disease <*S,
I was absent in all patients, as judged from the history and the physical |1
?• examination. • , ^<
I From the remaining 36 patients (table VI.l) two died within the study p
h period, and 17 of the 34 survivors had an uncomplicated AMI. A |';
•;', complicated AMI was characterized by the occurrence of pump failure
| (grade> II according to Killip and Kimbal 1967), or disturbances of cardiac j
| rhythm and conduction (excluding sinus bradycardia, sinus tachycardia,
I and extrasystolic beats). The hospital arrival time, defined as the time
interval between the acute onset of precordial pain and the time of
admission, was assessed in all patients. In two patients (nr. 2 and 17) the
peak SGOT value was found in the first blood sample withdrawn on ;
admission.
The treatment of the patients with AMI consisted in general of narcotic : ,
)- analgesics according to need, antiarrhythmic drugs (lidocaine for 36 hours '
! by intravenous drip, unless contraindicated), and anticoagulants (either j
continuation of coumarin derivatives in 22 patients, or institution of j
subcutaneous low-dose heparin in 12 patients); digoxin and diuretics were
prescribed, if necessary. Mobilization was begun on the third day after
admission, if feasible, and the administration of subcutaneous heparin was
discontinued some days later.
Blood samples were taken on days 1,2,3,4,5,6,7,10 and 14, between 0830
and 1030 a.m. except on day 1, when blood was taken at the time of
admission. In each plasma sample the following determinations were
done: TSH, rT3, T3, T4 and T3U. The competitive protein binding method
of Murphy and Pattee was used for the assay of T 4 . The 45 samples of 5
patients were also analyzed by T4-RIA, and the results were in close
agreement with the T4-D values. All samples of one patient were assayed in
the same run in order to avoid inter-assay variation. Hematocrit values
(Ht) were determined on the day of admission and in the second week.
Statistical evaluation of the trend in hormone levels was performed by
analysis of variance (Bennett and Franklin 1954). Differences between .
[ several points of time or groups of patients were analyzed by the (paired) t
I test. Because of their skewed distribution, plasma-TSH levels and the
| ratio's of plasma iodothyronine concentrations were analyzed after
I logarithmic transformation. In the statistical analyses the level of
' significance was taken as a = 0.05; undetectable hormone concentrations )
\ were taken as 50% of the detection limit. I

I VI. 3. Results. \
t i
I In the patients who died, a progressive decrease in plasma T3 and an ;
 143
0.8

0.6

0.4

0.2

i.i
2.4
G
I: ao
1.6

1.2

150

S 130

140

1 120 - — 1

u. 100

80

3.0

20

•5 — - — .

E 1.0
I
CO /
1-

0.5

0.3
1
w
days
Figure VIA.
Thyroid function tests (values as x ± SD but TSH as geometric mean ± SD) in 34patients with
acute myocardiai infarction.
Thyroid function tests in 34 patients admitted because of acute myocardial infarction (uncomplicated in 17 patients, group A; complicated in 17
patients, group B). Values as mean ± SD, except plasma TSH and iodothyronine ratio's which are presented as the geometric mean ± SD.
group dayl 2 3 4 5
TSH mU/1 all 0.9( 0 . 3 - 2 . 6 ) 0.7(<0.3 - 2.0) 1.0(0.4 - 2.7) 1.3(0.6 - 3.0) 1.35(0.6 - 2.8)
A 0.8«0.3 - 2.4) 0.7(<0.3 - 2.2) 1.1(0.5 - 2.7) 1.3(0.6 - 2.8) 1.3 (0.6 - 2.9)
B 1.1( 0.4-2.9) 0.7(<0.3 - 1.8) 0.9(0.3 - 2.7) 1.4(0.6 - 3.2) 1.4 (0.7 - 2.8)
rTa nmol/1 all 0.33 ± 0.08 0.47 ± 0.18 0.51 ±0.22 0.47 ± 0.16 0.44 ±0.15
A 0.32 ± 0.08 0.41 * 0.12 0.44 ± 0.14 0.41 ± 0.14 0.38 ± 0.13
B 0.33 ± 0.09 0.53 ± 0.21 0.58 ± 0.27 0.52 ± 0.17 0.50 ± 0.15
T3nmol/1 all 1.91 ± 0.36 1.62 ± 0.39 1.55 ± 0.39 1.65 ± 0.39 1.65 ± 0.37
A 1.90t 0.44 1.66 ± 0.42 1.67 ± 0.37 1.79 ±0.36 1.81 ± 0.33
B 1.91 ± 0.29 1.58 ± 0.36 1.43 ± 0.38 1.52 ± 0.38 1.49 ± 0.36
T« nmol/1 all 117 ± 19 117 ±22 119 ± 26 126 ± 29 128 ± 26
A 116 ±19 117 ± 24 123 ±28 129 ± 29 134 ±27
B 117 ±20 117 ±20 115 ±24 123 ± 31 121 ± 24
T3U all 0.94 ±0.11 0.96 ±0.12 0.95 ± 0.09 0.93 ± 0.08 0.93 ± 0.07
A 0.93 ± 0.08 0.95 ± 0.09 0.94 ± 0.06 0.91 ± 0.07 0.90 ± 0.06
B 0.96 ± 0.13 0.97 ± 0.14 0.96 ±0.12 0.95 ± 0.09 0.95 ± 0.08
FT4 index all 110 ±26 112 ±23 112 ± 24 117 ±28 118 ±23
A 108 ± 17 110 ±20 115 ±26 118 ±28 121 ±25
B 113 ±33 114±26 109 ± 22 116 ±30 115 ±21
rT 3 /T 3 xl0- 3 all 168(121 - 234) 282(193-413) 317(205 - 491) 277(179-429) 261(168 - 405)
A 167(115 - 244) 249(182 - 340) 259(180 - 373) 225(154 - 329) 205(141 - 299)
B 170(128 - 224) 320(212 - 483) 388(256 - 588) 341(228 - 508) 331(228 - 481)
rT 3 /T 4 xl0- s all 2.73(2.07 - 3.61) 3.85(2.78 - 5.35) 4.10(2.83 - 5.94) 3.62(2.57 - 5.10) 3.35(2.36 - 4.78)
A 2.69(2.03 - 3.58) 3.48(2.72 - 4.44) 3.51(2.60-4.76) 3.12(2.46 - 3.97) 2.78(2.11 - 3.66)
B 2.77(2.09 - 3.68) 4.27(2.94 - 6.21) 4.79(3.30 - 6.97) 4.20(2.89 - 6.09) 4.05(2.92 - 5.61)
T 3 /T 4 xl0" 3 all 16.2(13.3 - 19.8) 13.7(11.3- 16.4) 12.9(10.7 - 15.6) 13.1(11.0 - 15.5) 12.9(10.9 - 15.2)
A 16.1(13.2 - 19.7) 14.0(11.4-17.1) 13.6(11.3- 16.3) 13.9(11.4- 16.9) 13.5(11.2-16.3)
B 16.4(13.4-20.0) 13.4(11.3- 15.8) 12.3(10.2 - 14.9) 12.3(11.0-13.8) 12.2(10.7 - 14.0)
 group day 6 7 10 14 normal values
TSH mU/1 all 1.2(0.5 - 2.9) 1.1(0.5 - 2.6) 0.9( 0 . 3 - 2 . 2 ) 0.?( 0.3-2.5) 0.7(<0.3 - 2.3)
A 1.0(0.3 - 3.0) 0.9(0.3 - 2.6) 0.6«0.3 - 1.7) 0.7(<0.3 - 2.3)
B 1.4(0.7 - 2.6) 1.3(0.7 - 2.4) 1.2( 0.6-2.4) 1.2( 0 . 5 - 2 . 6 )
rT3 nmol/1 all 0.39 ±0.11 0.36 ± 0.10 0.33 ± 0.10 0.32 ±0.11 0.24 ± 0.07
A 0.36 ±0.11 0.33 ±0.11 1.30 ± 0.10 0.31 ± 0.09
B 0.42 ±0.10 0.38 ± 0.10 0.36 ±0.10 0.34 ± 0.12
T3 nmol/1 all 1.68 ± 0.39 1.62 ± 0.38 1.59 ± 0.28 1.63 ±0.32 1.85 ± 0.37
A 1.81 ± 0.37 1.76 ± 0.33 1.71 ± 0.19 1.69 ± 0.25
B 1.54 ± 0.36 1.49 ± 0.38 1.47 ± 0.31 1.57 + 0.37
T4 nmol/1 all 130 ±24 130 ±24 123 ±22 118 ±20 102 ± 18
A 134 ±25 130 ± 27 123 ± 19 117 ± 18
B 126 ± 22 129 ± 23 124 ± 25 119 ± 22
T3U all 0.93 ± 0.07 0.93 ± 0.07 0.93 ± 0.07 0.93 ± 0.08 0.94 ± 0.09
A 0.91 ± 0.05 0.92 ± 0.05 0.93 ± 0.05 0.92 ± 0.07
B 0.94 ± 0.09 0.94 ± 0.08 0.93 ± 0.09 0.94 ± 0.10
P\\ index all 120 ± 21 121 ±23 115 + 20 109 ± 18 96 ± 15
A 121 + 23 • 120 ± 25 114 ± 19 107 ± 17
B 118 ± 19 122 ± 22 115 + 21 111 ± 19
rT 3 /T 3 xl0- 3 all 229(155 - 337) 224(142 - 352) 203(133- 309) 191(122 - 300) 124(86 - 179)
A 193(133-280) 183(126 - 264) 170(115-252) 176(120 - 257)
B 271(195 - 376) 274(176 - 428) 243(167 - 354) 209(126 - 347)
rT 3 /T 4 xl0" 3 all 2.91(2.16 - 3.92) 2.69(2.03 - 2.56) 2.62(1.90 - 3.60) 2.62(1.90 - 3.62) 2.23(1.62 - 3.08)
A 2.60(2.01- 3.37) 2.47(1.92 - 3.18) 2.38(1.76 - 3.22) 2.53(1.88 - 3.41)
B 3.26(2.43 - 4.38) 2.93(2.19 - 3.90) 2.88(2.10 - 3.94) 2.71(1.91 - 3.85)
T 3 /T 4 xl0" 3 all 12.7(10.6 - 15.4) 12.4( 9.7 - 15.7) 12.9(10.6 - 15.6) 13.7(11.0- 17.0) 18.0(14.9 - 21.9)
A 13.5(11.0 - 16.5) 13.5(10.9 - 16.7) 14.0(11.8-16.6) 14.4(11.7 - 17.8)
B 12.1(10.3 - 14.1) 11.3( 8.9 - 14.2) 11.9( 9.9 - 14.2) 13.0(10.4 - 16.2)
 146
Table Vl-3
Plasma T 3 and rT3 concentrations (values as mean ± SD) on admission as a function of
ft
f.
the hospital arrival time (HAT) in 34 patients with acute myocardial infarction.

HAT n T3 *T3
hrs. nmol/1 nmol/1

< 2 8 1.94 ± 0.39 0.29 ± 0.05

2-3 S 1.98 ± 0.31 0.31 ± 0.05
3-4 4 1.89 ± 0.24 0.30 ± 0.05
4-S 7 1.82 ± 0.37 0.30 ± 0.12
5-6 3 1.89 ± 0.46 0.34 ± 0.06
6-24 5 2.11 ± 0.34 0.39 ± 0.07
> 24 2 1.45 ± 0.49 0.47 ± 0.12

increase in plasma rT3 levels was observed. The results of the thyroid
function tests in the 34 survivors are presented in figure VI. 1. and table
VI.2. A transient increase in the plasma levels of TSH, rT3 and T4 (p <
0.001) and in the FT4 index (p < 0.01) was found, whereas plasma T3 (p <
0.001) and T3U (p< 0.05) decreased. No difference was found in the plasma
concentrations of TSH, rT3 and T4 and in the FT4 index between days 1
and 14, but plasma T3 and T3U were lower on day 14. The trend in
hormone levels in the patients with an uncomplicated AMI (group A) was
similar to the trend found in those with a complicated AMI (group B). The
increase in plasma rT3 and the decrease in plasma T 3 was greater in group
A than in group B, but plasma TSH, T4 and T3U and the FT4 index did
not differ between these two groups. Peak SGOT values were higher in
complicated AMI than in uncomplicated AMI (p < 0.01). Concomitant
with the changes in the plasma iodothyronine concentrations a transient
increase in the ratio's of rT 3 /T 3 and rT3/T4 (p. < 0.001) and a decrease in
the ratio of T 3 /T 4 was found (p < 0.001). The return to base-line values of
the ratio's was delayed in group B as compared with group A. No
difference was found between day 1 and day 14 in the ratio's of rT3 / T3 and of
rT3/T4, but the ratio T 3 /T 4 was lower on day 14 than on day 1 ( p < 0.01).
The 34 patients were further divided in three groups according to infarct
size as estimated by the peak SGOT value (table VI.4). The increase in
plasma rT3 and the decrease in plasma T3 concentrations (figure VI.2) were
calculated as the difference between the highest or lowest observed value
and the value on day 14, since the initial values of these hormones on the
day of admission were clearly related to the hospital arrival time (table
VI.3). No differences were found in any of the parameters tested between
patients with small infarctions (peak SGOT < 80) and those with medium
sized infarctions (peak SGOT 80-160). However, the values of A rT3, lowest
T3 and A T3, and highest rT 3 / T3 and rT3 / T4 of patients with large infarctions
 147
Table VI-4
Some parameters of thyroid hormone metabolism (values as x ± SD, ratio's as geometric
mean ± SD) in 34 patients with acute myocardial infarction, divided in three groups
according to infarct size as estimated by the peak SGOT value.

peak SGOT value

<80 80 - 160 >160
(n=14) (n=12) (n=8)

peak SGOT 52 ± 1 6 121 ± 23 205 ± 36

highest rT3 0.53+ 0.19 0.56+ 0.18' 0.65 ± 0.37'
ArT3° 0.17 ± 0.14 0.24+ 0.15' 0.39 ± 0.34 3
lowest T3 1.37+ 0.19 1.31 ± 0.351 1.16 ± 0.23 3
AT3° 0.16+ 0.30 0.36+ 0.28 2 0.60 ± 0.20 4
highest rT3/T3 339(237 -486) 357(231 - 552)' 478(346 - 660) 3
highest rT3/T4 4.12( 2.98 -5.71) 4.88( 3.41 -6.98)' 5.48( 4.14-7.25) 3
lowest T 3 /T 4 10.9 ( 9.3 -12.9) 11.1 ( 9.0 -13.6)' 10.2 ( 9.2 -11.4)'

°ArT 3 andAT 3 : the difference between the highest or lowest observed value and
the value on day 14.
1
not significant;2 0.05<p<0.1; 3 p<0.05; * p<0.01, as compared with the values of the
'peak SGOK80' group.

(peak SGOT > 160) were different from those of patients with small
infarctions. The peak values of T4 and TSH did not differ in these three
groups, although a tendency to higher peak TSH values was observed in
patients with greater infarctions. A linear correlation was found between the
infarct size and all of the following parameters: A rT3, AT3, highest rT 3 /T 3

Table VIS
The correlation between infarct size as estimated by the peak SGOT value (variable x) and
some parameters of thyroid hormone metabolism (variable y) in 34 patients with acute
myocardial infarction.

variable y regression equation coefficient p-value

of correlation

highest rT3 y = O.OOlx + 0.46 r=0.28 0.05<p<0.1

ArT3 y = O.0015x + 0.07 r = 0.46 p<0.01
lowest T 3 y=-O.OOlx + 1.43 r = 0.28 0.05<p<0.1
AT3 y = 0.003x + 0.03 r = 0.56 p<0.001
highest rTj/Tj y = O.96x + 293.56 r=0.36 p<0.05
highest rT3/T4 y = 0.01x + 3.84 r = 0.34 p<0.05
lowest T 3 /T 4 y =-0.004x + 11.35 r = 0.12 N.S.
I-
3

148
PEAK SGOT (U/l)
<80 80-160 >160
0.75 r (n=14) (ru8)

So 0-50

0.25

0.25
S
o
c

0.50

L
0.75

Figure VI.2.
The increase in plasma rTj and the decrease in plasma T3 concentrations in 34 patients with
acute myocardial infarction, divided according to infarct size as estimated by the peak
SGOT value. ( AIT, and A T3: the difference between the highest or the lowest observed value
and the value on day 14; values as x±SEM).

and highest rT3/T4 (table VI.5 and figure VI.3). As depicted in figure VI. 1,
the peak values of TSH precede those of T4 in time. Inspection of thedata of
each individual strongly suggested this sequence in most cases. Since the day
on which the peak T4 value was reached, differed f rompatient to patient, we
rearranged the T4 values according to that day. The TSH- and T3-values
were similarly rearranged around the peak T4 value. Isolated high T4 values
found on occasion during the first three days after AMI, were not considered '•4-
as the "peak", since they apparently were due to the initial hemoconcentra-
tion: Ht values on admission were higherthan in thesecond week (0.45±0.04
 ^ _^ ,„ .,„,,,, -,-s.r:7---v^fJ*«J2^S^lt?*#^?'"r .'?-S'f\'^s"lT''$'?t'?''.*l"'-» '.'

Table VT-6
Plasma T 4 , TSH and T, concentrations in 34 patients with acute myocardial infarction, arranged around the time of the observed "peak" T,
value in the first two weeks after admission (T4 and T3 values as x ± SD with the range in brackets, TSH as geometric mean ± SD).

day -7 -6 -5 -4 -3 -2
n (6) (13) (22) (28) (33) (34)

f4 nmol/1 108 ± 21 101 ± 20 109 ± 19 113 ± 21 119 ± 22 125 ± 25

( 8 6 - 138) ( 7 9 - 144) ( 7 2 - 138) ( 7 2 - 150) ( 8 0 - 173) ( 8 9 - 188)
TSH mU/1 0.6(<0.3 - 2.1) 0.7(<0.3 - 3.0) 0.6«0.3 - 2.0) 0.7(<0.3 - 1.9) 1.3(0.6 - 3.0) 1.3(0.7 - 2.5)
T3 nmol/1 1.59 t :0.31 1.70:t0.52 1.54 ± 0.47 1.54:tO.36 1.62 ± 0.38 1.68 d:0.35
(1.03 - 1.94) (0.85 - 2.63) 0.89- - 2.36) (0.91 - 2.42) (0.99 - 2.53) (0.92 - 2.39)
4
- peakT4
day —1 0 +1 +2 +3 +4 +5
n (34) (34) (33) (28) (21) (12) (6)
T4 nmol/1 129 ± 24 139 ± 26 129 ± 22 123 ± 23 126 ± 20 123 ± 25 127± 26
(93 - 182) ( 9 7 - 197) ( 9 7 - 187) ( 8 5 - 169) ( 9 2 - 178) ( 9 4 - 159) ( 9 0 - 147)
TSH mU/1 1.4(0.7 - 2.9) 1.2(0.5 - 2.7) 1.1(0.4 - 3.1) 0.9(0.4 - 2.2) 1.0(0.4 - 2.6) 0.9(0.4 -2.0) 1.0(0.4 - 2.5)
T3 nmol/1 1.68 ± 0.33 1.79 ± 0.42 1.67 ± 0.35 1.67 ± 0.27 1.67 ± 0.35 1.61 ± 0.29 1.53:t0.51
(0.99 - 2.22) (1.00 - 2.63) (1.14 - 2.43) (1.23 - 2.34) (0.80 - 2.16) (0.85 - 1.94) (0.71 - 2.20)
 150
r=0.46 1.2 r=0.56
1.2 p<0.001
p<0.01

1.0 1.0

|0.8 0.8
"rii-
3
50.6 10.6

0.4

0.2 0.2

40 80 120 160 200 240 40 80 120 160 200 240

PEAK SGOT U/l PEAK SGOT U/l

g F/.5.
The relation between the changes in the conversion of T4 (as indicated by A rT3 and &T3: the
difference between the highest or lowest observed value and the value on day 14) and the
infarct size (as estimated by the peak SGOT value) in 34 patients with acute myocardial
infarction.

; •
vs. 0.43±0.04, p < 0.001). The results are listed in table VI.6. Again, the
substantial increase in T4 is preceded by a rise in TSH, and is associated
with a concomitant increase in plasma T3 values.

VI. 4, Discussion.

The reciprocal changes in plasma concentrations of T3 and r T3 found in

our patients with acute myocardial infarction, are similar to those found in
other acute non-thyroidal diseases (see chapter V). The decrease in plasma
T3 and the increase in plasma rT3 thus seems to be caused by inhibition of
the 5'-deiodinating process in the peripheral conversion of T4. The nadir of
T3 and the peak of rT3 occurred on the third day, in agreement with the
study of Westgren et al (1977). As in patients with other non-thyroidal
diseases, the decrease in plasma T3 in patients with AMI is not related to
the increase in plasma cortisol levels (Ljunggren et al 1979).

A qualitative relationship between the severity of AMI and the changes in

the conversion of T4 was found: in patients with a complicated AMI the
decrease in plasma T3 and the increase in plasma rT3 was greater than in
patients with uncomplicated AMI, and the return to base-line levels was
delayed in complicated AMI as compared to uncomplicated AMI. The
greatest fall in plasma T3 in patients with the most severe course of AMI
has also been observed by Naumann et al (1975). Our data also indicate
that there is a quantitative relationship between the severity of AMI and
&*.' 151
the changes in the conversion of T4, since the infarct size was greater in
patients with a complicated AMI, as was expected (Chapman 1972). The
existence of a quantitative relationship is further demonstrated by the
linear correlation between peak SGOT values and various parameters of 8
thyroid hormone metabolism: A rT3, AT3,'highest rT3 / T3 and rT3 / T4 ratio's
(but not with the lowest T3/T4 ratio). The only report in the literature
relating infarct size to thyroid hormone metabolism is of Smith et al
(1978). They found a quantitative relationship between the infarct size and
the lowest T3/T4 ratio (but not with the lowest plasma-T3 value). The cause
for the discrepancy in results is probably related to a different selection of
patients: we excluded only those patients who were treated with drugs that
interfere with thyroid function tests, whereas Smith et al (1977) applied
more rigid selection criteria.
A real quantitative relationship between the severity of a disease and the
changes in thyroid hormone metabolism in other states than AMI has thus
far only been reported in diabetes (Saunders et al 1978; Pittman et al 1979).
A negative correlation between fasting blood glucose levels and the T3/T4
ratio has been demonstrated in diabetic patients, and plasma T3 was
positively related to glucose utilization and glucose MCR but negatively to
plasma ketone body concentration.

As in other non-thyroidal diseases, the fate ofthyroxine in patients with

AMI is most intriguing. The occasionally found high T4 values on the first
three days are best explained by hemoconcentration, as witnessed by the
observed higher hematocrit values at the time of admission, and the
reported high Ht values in the first days after the onset of AMI followed by
a steady decrease up to day 10 (Jan et al 1975; Kaplan et al 1977; Smith
1977). Vanhaelst et al (1976) have suggested that the elevated plasma T4
levels at the onset of AMI are due to stimulation of thyroid secretion
induced by the rise in catecholamines, which can stimulate thyroid
secretion in animals (Ericson et al 1970; Melander 1970). This explanation
seems, however, rather far-fetched in the light of the plausible explanation
of hemoconcentration. Also, the thyroxine secretion rate determined
immediately after AMI has been reported as normal (Harland et al 1972).
One would expect a decrease in plasma T4 values concomitant with the
decrease in Ht (Kaplan et al 1977). Smith et al (1978) found no changes in
the T4 values (which therefore already indicates an increase in circulating
T4), but after correction for the change in Ht an increase of 15% in T4 levels
between day 2 and day 9 was calculated (Smith 1977). We observed a clear
increase in uncorrected plasma T4 values of appr. 12%, peaking around
day 6-7 (table VI.2). This increase is not caused by hemoconcentration,
since the Ht decreased and T3U remained constant as of day 3. The initially
higher T3U values - and not lower values as would be expected from the
hemoconcentration in the initial phase (cf. table 11.21) - are most probably
 152
caused by a decreased binding capacity of the plasma proteins for thyroid
hormones, analogous to the situation found in other non-thyroidal
diseases (see chapter VI), due to a decrease in TBP caused by the 'acute
phase reaction' (Johansson et al 1972; Smith et al 1977; Kushner et al
1978). The concentration of TBG has been measured in patients with AMI, I
and after an initial fall to a nadir on day 3 a return to the initial levels is
! - •
observed (Westgren et al 1977). Thus the increase in T4 is not due to
changes in the plasma volume or to changes in the concentration of thyroid
hormone binding proteins.
It is also highly unlikely that the increase in T4 is caused by a rise in
the plasma concentration of free fatty acids (FFA) for several
reasons:
1) FFA falsely increase T4-values as measured in the competitive-protein
binding method of Murphy and Pattee (T4-D assay), but are without
effect on the results in theT4-RI A (Rootwelt 1975; Liewendahl and Hele-
nius 1976; Pannal et al 1977); the rise of T4 in our study was demonstrated
byT 4 -DandbyT 4 -RIA.
2) intravenous heparin increases T4 (as measured in theT4-D assay) and FT4

s
(Saeed-Uz Zafar et al 1971), most probably via the induced rise in FFA;
subcutaneous low-dose heparin does not produce a rise in FFA
(Arnesen et al 1979), and furthermore the increase in T4 was also seen in
our patients who were treated with oral anticoagulants, which are with-
out effect on T4 and FT4 concentration in AMI (Saeed-Uz Zafar et al
1971).
3) the catecholamine-mediated rise in FFA in AMI is an early phenome-
non, the highest levels of FFA are found at the time of admission where-
after a decrease to near normal levels on day 3-5 occurs (Gupta et al 1969;
Sharma 1977); however, plasma T4 peaks later, on day 6-7.
It thus appears that there is a real increase in the amount of circulating
T4 around day 6-7 - in fact, a laboratory pattern of "T4-toxicosis" is found
in 16 of the 34 patients at the time of the T4 peak. In general, an increase in
plasma T4 is caused by either a decrease in MCR or an increase in PR. The
impairment in the deiodination of T4 (which is quantitatively an important
pathway in the degradation of T4) may have lowered the M CR. A higher P R
is caused by increased thyroidal secretion of T4. Since the T4 peak is
preceded in time by a significant increase in TSH, this suggests that a TSH-
mediated secretion of T4 is responsible (at least in part) for the rise in T4.
Furthermore, indirect evidence for this sequence of events is found in the
associated rise of plasma T3 (table VI.6).
If the T4 rise is TSH mediated, the question arises why the release of
TSH is stimulated. Again, the time sequence suggests that the low T3 levels
play a causative role - mean lowest T3 levels are found on day 3, mean
highest TSH levels on day 4 and 5. In the rat, TSH release by the anterior
pituitary is a function of nuclear T3, which is derived half from
 153
intrapituitary T4 -*T3 deiodination and half from plasma T3 (Silva and
Larsen 1978). A normal concentration of FT4 is reported in the initial
phase of AMI, whereas a substantial decrease in FT3 is observed starting 8
hours after the onset of AMI (Saeed-Uz Zafar et al 1971; Bugiardini et al
1979). Therefore, it seems reasonable to propose that the fall in T3 and FT3
concentrations provokes the release of TSH which, in a later phase, is
switched off by the increase in T4.

In summary, our studies suggest the following sequence of events in

thyroid hormone metabolism in patients with AMI:
1. the occasionally observed high T4 values in the initial phase are due to
hemoconcentration.
2. the 5'-deiodination of T4 is inhibited, resulting in a peak of plasma rT3 and
in a nadir of plasma T3 values on day 3, and in a lower metabolic clearance
ofT4.
3. TSH secretion, provoked by the lower T3 levels, increases and JV-AICS on
days 4 and 5.
4. a TSH-mediated increase in thyroidal secretion of T4 and T3 occurs
mainly on days 6 and 7, and is switched off thereafter by the negative
feedback of thyroid hormones on the pituitary.
5. subsequently, all parameters return to normal in about two weeks with
the exception of the T4-*T3 deiodination which remains impaired up to
appr. 3 months after AMI (Smith et al 1978).
This proposition satisfactorily explains many of the seemingly
contradictory results of thyroid function tests in AMI so far published.

From a teleological point of view, the low T3 levels in AMI may serve to
prevent further catabolism (see chapter IX). In this respect the observed
changes in thyroid hormone metabolism in patients with AMI fits in the
general adaption syndrome of the body to stress. It remains to be
demonstrated if it is wise to treat the increase in thyroidal secretion with
antithyroid drugs as has been proposed (Ceremuzynsky et al 1970). On the
other hand, recent studies on the allegedly beneficial effect of B-
adrenoceptor blocking-agents in the reduction of infarct size (Julian 1976;
Hjalmarson and Werko 1976) are of great interest, since one of the effects
of these drugs is impairment of the S'-deiodination of T4 (cf. chapters VII
and VIII).

VI.5. References

Arnesen H, Bjerkedal I, Skjaeggestad 0 , Godal HC. Plasma free-fatty-acids and small doses
of subcutaneous heparin in acute myocardial infarction. Thrombosis Research 1979; 14:
541-9.
 •WJ-

154
Bennett CA, Franklin NL. Statistical analysis in chemistry and the chemical industry. New
York: John Wiley and Sons, 19S4.
Bugiardini G, Miniero R, Mattioli L et al. Modificazioni di ormoni tiroidei in corso di
infarcto miocardico acuto. In: The Proceedings of the International Symposium on free
thyroid hormones (Venice 1978). Amsterdam: Excerpta Medica, 1979 (in press).
Ceremuzynski L, Kuch J, Markiewicz L, Lawecki J, Taton J. Patterns of endocrine reactivity
in patients with recent myocardiat infarction. Clinical and biochemical correlations: trial
of endocrine therapy. Brit Heart J 1970; 32:603-10. '€••
Chapman BL. Relation of cardiac complications to SGOT level in acute myocardial
infarction. Brit Heart J 1972; 34:890-6.
The Criteria Committee of the New York Heart Association. Nomenclature and criteria
for diagnosis of diseases of the heart and great vessels. 7th ed. Boston: Little, Brown and
Compagny, 1973.
Ericson LE, Melander A, Owman Ch, Sundler F. Endocytosis of thyroglobulin and release of I:
thyroid hormone in mice by catecholamines and 5-hydroxy-tryptamine. Endocrinology
1970:87:915-21.
Gupta DK, Young R, Jewitt DE, Hartog M, Opie LH. Increased plasma-free-fatty-acid con-
centrations and their significance in patients with acute myocardial infarction. Lancet
1969; 2:1209-13.
Harland WA, Orr JS, Dunnigan MG, Sequeira RFC. Thyroxine secretion rate after
myocardial infarction. Brit Heart J 1972; 34: 1072-4.
Hjalmarson A, Werko L (editors). Experimental and clinical aspects on preservation of the
ischetnic myocardium. Acta Med Scand 1976; suppl 587:177-220.
Jan KM, Chien S, Bigger JT. Observations on blood viscosity changes after acute myocardial
infarction. Circulation 1975; 51:1079-84.
Johansson BG, Kindmark CO, Trell EY, Wollheim FA. Sequential changes of plasma
proteins after myocardial infarction. Scand J Clin Lab Invest 1972; 29: suppl 124,117-26.
Julian DG (chairman). Beta-blockade and myocardial infarction. Postgrad Med J 1976; 52:
suppl 4:124-56.
Kaplan MM, Schimmel M, Utiger RD. Changes in serum 3,3',5'-triiodothyronine (reverse
T3) concentrations with altered thyroid hormone secretion and metabolism. J Clin Endo-
crinol Metab 1977; 45:447-56.
Kibe O, Nilsson NJ. Observations on the diagnostic and prognostic value of some enzyme
tests in myocardial infarction. Acta Med Scand 1967; 182:597-610.
Killen DA, Tinsley EA. Serum enzymes in experimental myocardial infarcts. Relation of
blood levels of serum glutamic oxaloacetic transaminase, lactic dehydrogenase, and heat
stable lactic dehydrogenase to size of experimental myocardial infarct. Arch Surg 1966;
92:418-22.
Killip T III, Kimbal JT. Treatment of myocardial infarction in a coronary care unit: a two
year experience with 250 patients. Am J Cardiol 1967; 20:457-64.
Kushner I, Broder ML, Karp D. Control of the acute phase response. Serum C-reactive
protein kinetics after acute myocardial infarction. J Clin Invest 1978; 61:235-42.
Ljunggren J-G, Falkenberg C, Savidge G. The influence of endogenous cortisol on the
peripheral conversion of thyroxine in patients with acute myocardial infarction. Acta Med
Scand 1979; 205:267-9.
Liewendahl K, Helenius T, Effects of fatty acids on thyroid function tests in vitro and in
vivo. Clin Chim Acta 1976; 72:301-13.
Melander A. Amines and mouse thyroid activity: release of thyroid hormone by catechol-
amines and indoleamines and its inhibition by adrenergic blocking drugs. Acta Endo-
crinol 1970; 65:376-80.
Nauman J, Ceremuzynski L, Nauman A, Gunther-Krawezynska E, Jarczynska J. Serum
triiodothyronine, thyroxine and thyroid stimulating hormone in relation to the clinical
 155

I course of recent myocardial infarction. Abstract 94, 9th annual meeting European
Society for Clinical Investigation. Rotterdam 1975.
Pannall PR, Swanepoel E, Bennett JM, Pauw FH. Increased concentrations of serum free
fatty acids falsely increase serum thyroxine as determined by competitive protein-binding.
ClinChem 1977; 6:990-3.
Pittmann CS, Suda AK, Chambers JB Jr, Ray GY. Impaired 3,5,3'-triiodothyronine (T3)
production in diabetic patients. Metabolism 1979; 28:333-8.
Rootwelt K. The influence of fatty acids on serum thyroxine determinations by competitive
protein-binding assay. Scand J Clin Lab Invest 197S; 35:649-53.
Saeed-Uz Zafar M, Miller JM, Breneman GM, Mansour J. Observations on the effect of
heparin on free and total thyroxine. J Clin Endocrinol Metab 1971; 32:633-40.
Saunders J, Hall SEH, SOnksen PH. Thyroid hormones in insulin requiring diabetes before
and after treatment. Diabetologia 1978; 15:29-32.
Sharma SC. Catecholamines and free fatty acids in myocardial infarction and angina. J Clin
Pathol 1977; 30:1037-9.
Silva JE, Larsen PR. Contributions of plasma triiodothyronine and local thyroxine mono-
deiodination to triiodothyronine to nuclear triiodothyronine receptor saturation in
pituitary, liver and kidney of hypothyroid rats. Further evidence relating saturation of
pituitary nuclear triiodothyronine receptors and the acute inhibition of thyroid-stimu-
lating hormone release. J Clin Invest 1978; 61:1247-59.
Smith SJ, Bos G, Esseveld MR, van Eyk HG, Gerbrandy J. Acute-phase proteins from the
the liver and enzymes from myocardial infarction; a quantitative relationship. Clin Chim
Ada 1977; 81:75-85.
Smith SJ. Plasma-eiwitten en collold-osmotische druk na het acute myocard infarct. Thesis
Rotterdam 1977.
Smith SJ, Bos G, Gerbrandy J, Docter R, Visser TJ. Lowering of serum 3,3',5-triiodo-
thyronine thyroxine ratio in patients with myocardial infarction: relationship with extent
of tissue injury. Eur J Clin Invest 1978; 8:99-102.
V&nhaelst L, Degaute JP, Golstein J, Bastenie PA. Pituitary-thyroid axis reaction after
myocardial infarction. Horm Metab Res 1976; 8:42-6.
West M, Eshchar J, Zimmerman HJ. Serum enzymology in the diagnosis of myocardial infarc-
tion and related cardiovascular conditions. Med Clin N Am 1966; 50: 171-92.
Westgren U, Burger A, Levin K, Melander A, Nilsson G, Petterson U. Divergent changes of
serum 3,5,3'-triiodothyronine and 3,3',5'-triiodothyronine in patients with acute
myocardial infarction. Acta Med Scand 1977; 201:269-72.
 V

CHAPTER VII.
STUDIES ON THE EFFECT OF PROPRANOLOL IN HUMAN
SUBJECTS

VII. 1. Introduction.
Although it is well known that in thyrotoxic patients treatment with a 0-
adrenoceptor blocking agent will reduce many of the manifestations of the
disease (Shanks et al 1969; Malcolm 1972), the precise mode of action of
these agents in hyperthyroidism is not well understood (Levey 1971;
Spauldingand Noth 1975; Levey 1975;Landsberg 1977). Since propranolol
does not decrease plasma thyroxine (T4) or "'I-uptake in the thyroid
(Krikler 1966; Hadden et al 1968,1969; Biran and Tal 1972; McLarty et al
1973) it is thought that 0-blocking agents do not act directly on the
hyperactive thyroid, but rather decrease the — perhaps increased —
sensitivity of the|3-adrenoceptors in the hyperthyroid patient.
The possibility that0 -blocking agents influence the p!asma concentra-
tion of triiodothyronine (T3) suggested itself in a goitrous patient, who was
under treatment with alprenolol (400 mg per day) because of vague cardiac

vulhyroid euthyraid

\, 1
'1

20
> day»

Figure VIM. Effect of alprenolol treatment on plasma levels of T4, T, (solid lines) and rT,
(dotted line) in a patient with multinodular goitre. (TSH-Ievels were below the detection limit
of the assay during the whole period depicted; the TSH-response to TRH, days 0,20 and 42,
was absent).
 157
£? complaints. Although this 59-year old woman was clinically euthyroid, the
if
W:-
results of thyroid function tests were compatible with a diagnosis of
hyperthyroidism, except for the fact that plasma-T3 measured on several
i
JT occasions was either marginally elevated or even normal. Discontinuing
; the alprenolol medication led to a rise in plasma-T3 to clearly hyperthyroid
;•£ levels (figure VII. 1) and the patient became clinically thyrotoxic. When the
U treatment was resumed plasma-T3 decreased again to normal levels.
ft We therefore decided to study the effect of therapy with a 0-blocking agent
in hyperthyroidism on the plasma levels of T4, T3, rT3 and TSH. A group of
patients with primary hypothyroidism on substitution therapy with L-
thyroxine was included in the study in order to differentiate between a
thyroidal or extrathyroidal effect, if any. Propranolol was chosen because il
it is the best known0-antagonist and is widely used in the treatment of
hyperthyroidism (Ramsay 1975). Moreover, propranolol has no intrinsic
sympathomimetic activity.
This study has been published in part elsewhere (Wiersinga and Touber
1977).

VII.2. Patients and methods.

Group A consisted of 11 hyperthyroid patients (10 females, one male, I

varying in age from 22 to 73 years, with a mean of 54.8 years); six patients jr-
had Graves' disease, three had a toxic multinodular goitre and two a toxic
adenoma. In the hyperthyroid patients the mean T4 was 213 nmol/1, range
163-259, the mean T3 4.56 nmol/1, range 2.70-6.14. Of the hyperthyroid
patients six were newly diagnosed cases and had never been treated. Five
others had a relapse of thyrotoxicosis; in these patients no antithyroid
medication had been given for at least two months prior to the study. Two
of the five patients had been treated with radioactive iodine two and twelve
years before the study.
Group B consisted of six hypothyroid patients (all female, varying in age
from 33 to 68 years with a mean of 58.5 years); three patients had primary
myxoedema, one was hypothyroid after subtotal thyroidectomy and two
had undergone total thyroidectomy because of thyroid carcinoma
(follicular and papillary carcinoma, respectively). The last two patients
had a neck uptake of less than 0.4% of the administered dose of I31I two
months prior to the study; no signs of functioning metastases could be
II
detected by whole-body scintigraphy at that time. All six patients were on a
constant substitution dose of L-thyroxine (200//g per day in five and 150
I
it
Mg per day in one patient) for at least one month prior to the study and all
were clinically euthyroid, their mean T4 was 114 nmol/1, range 89-146;
mean T3 1.80 nmol/1, range 1.29-2.28.
Both groups of patients received a fourteen-day course of propranolol,
consisting of 80 mg per day in the first and 160 mg per day in the second
 Table VIM
Thyroid function tests during propranolol treatment, 80 mg/day in the first and 160 mg/day in the second week (values as x ± SD, but plasma
TSH and iodothyronine ratio's as geometric mean ± SD; day 0 represents the mean of the two days prior to treatment).
days 0 1 2 3 5
group A. hyperthyroid patients (n = 11)
"i TSH mU/1 <0.3 <0.3 <0.3 <0.3 <0.3
rT3 nmol/1 0.91 ± 0.43 0;97 ± 0.37 0.92 ± 0.37 1.03 ± 0.38 1.01 ± 0.42
Ts nmol/1 4.S6 ± 1.22 4.45 ± 1.29 4.05 ± 1.26 3.99 ± 1.26 3.91 ± 1.20
T4 nmol/1 213 ±32 213 ± 39 210 ± 39 212 ± 43 214 ± 40
T3U 1.16 ± 0.15
FT4 index* 2S0 ± 58
rT 3 /T 3 xl0- 3 189(126 - 284) 209(150 - 292) 221(152 - 322) 253(176 - 365) 248(153 - 403)
rT 4 /T 4 xl0" 3 3.95(2.76-5.64) 4.27(3.08-5.92) 4.13(2.97-5.74) 4.64(3.43-6.27) 4.41(3.03-6.40)
T 3 /T 4 xl0- 3 20,9(16.4-26.6) 20.4(16.6-25.1) 18.7(15.4-22.8) 18.3(15.0-22.3) 17.8(13.9-22.7) 00

group B. hypothyroid patients on L-T4 substitution (n = 6)

TSH mU/1 1.3(<0.3 - 9.1) 1.3«0.3 - 8.9) 1 . 6 « 0 . 3 - 11) 1.6«0.3 - 12) 1.7(<0.3 - 13)
rT3 nmol/1 0.22 ± 0.14 0.30 ± 0.20 o!31 ± 0.15 0.29 ± 0.12 0.26 ±0.11
T3 nmol/1 1.80 ± 0.37 1.93 ± 0.55 1.79 ± 0.54 1.65 ± 0.54 1.45 ± 0.42
T4 nmol/1 114 ± 21 114 ±23 115 ± 22 114 ±23 119 ±26
T3U 0.96 ± 0.08
FT4 index 108 ± 27
rT 3 /T 3 xl0" 3 105( 50 - 221) 131( 67 - 258) 165( 92 - 298) 173(102 - 294) 167( 86 - 323)
rT 3 /T 4 xl0" 3 1.64(0.83-2.27) 2.14(1.17-3.89) 2.52(1.58-4.01) 2.42(1.61-3.64) 2.00(1.39-2.89)
T 3 /T 4 xl0" 3 15.6(12.0-20.4) 16.6(12.0-23.0) 15.2(10.7-21.6) 14.0(10.1-19.4) 12.0( 8.4-17.2)
days 12 14
group A. hypeTthytoid patients (n = 11)
TSH mU/1 <0.3 <0.3 <0.3 <0.3
rT3 nmol/1 1.02 ± 0.30 1.04 ± 0.39 1.09 ± 0.39 1.12 ±0.44
T3 nmol/1 3.76 ± 1.09 3.68 ± 1.16 3.57 ± 1.08 3.20 ±1.16
T4 nmol/1 215+31 215 ± 37 218 ±40 208 ± 37
T3U 1.19 ± 0.19
FT4 index 6 250 ± 75
rT 3 /T 3 xl0 l 271(188 - 391) 276(176 - 432) 297(185 - 475) 343(216 - 543)
rT4/T4xl0"3 4.59(3.48-6.04) 4.59(3.41-6.16) 4.74(3.28-6.84) 5.05(3.46-7.36)
T 3 /T 4 xl0- 3 16.9(13:4-21.4) 16.6(13.2-20.9) 16.0(12.6-20.0) 14.7(11.4-19.0) in

group B. hypothyroid patients on L-T, substitution (n = 6)

TSH mU/1 1.9(0.3-13) 1.9(<0.3 - 17) 1.8(<0.3 - 17) 2.2(0.3 - 19)

rT3 nmol/1 0.28 ±0.11 0.30 ±0.18 0.28 ±0.13 0.34 ± 0.16
T3 nmol/1 1.48 ± 0.31 1.51 ± 0.46 1.42 ± 0.34 1.29 ± 0.42
T4 nmol/1 119 ±23 123 ±26 123 ± 26 120 ± 19
T3U 0.97 ± 0.06
FT4 index 117 ±25
rT3/T3xl0"3 174( 99 - 305) 175( 81 - 379) 176( 85 - 365) 245(118-509)
rT3/T4xl0"3 2.18(1.40-3.39) 2.13(1.20-3.78) 2.04(1.09-3.80) 2.55(1.53-4.28)
T 3 /T 4 xl0" 3 12.5( 9.3-16.9) 12.2( 8.9-16.7) 11.5( 8.7-15.2) 10.4( 8.1-13.3)
 160
week; other medication, if any, was kept constant during the study period.
Propranolol was administered 8-hourly in oral doses of 40-20-20 mg and
80-40-40 mg, respectively. Blood was taken 90 min after the morning dose;
the patients were not allowed to take any drug (including L-thyroxine)
except propranolol, before blood sampling was completed. Blood samples
t
were collected two days prior to, and on days 1,2,3,5,7,9,12 and 14 of the
propranolol medication. In each sample the following determinations
were done in plasma: T4 by the competitive protein binding method of
Murphy-Pattee, T3, rT3 and TSH by radioimmunoassay. Stimulation tests
with thyrotropin releasing hormone were carried out before and on the last
day of treatment (200 fig TRH through an indwelling venous catheter, in
the second test 90 min after 80 mg propranolol orally; TSH- and T3- i'
responses were followed for 4 hours). The triiodothyronine uptake ratio
(T3U) was measured before and on the last day of propranolol treatment.
All samples of one patient were run in the same assay in order to avoid
interassay variation. Pulse rate, blood pressure and weight of each patient

. I
were recorded on the same days as the blood samples were taken.
Statistical evaluation of the trend in hormone levels and of the TSH- and
T3-responses after TRH stimulation were performed by analysis of
1
variance (Bennett and Franklin 1954). Plasma-TSH levels and the ratio's
of iodothyronines, because of skewed distribution, were analyzed after
logarithmic transformation. Duncan's test (1955) was used to determine
the day on which the mean T3 value was different from the pre-treatment
value. T3U and the calculated FT4 index were evaluated by Student's
paired t test. In all analyses the level of significance was taken as a =0.05;
undetectable hormone concentrations were taken in the statistical analyses
as 50 percent of the detection limit.

VII.3. Results.

The results are presented in tables VII. 1 and VII.2. Propranolol

treatment of the hyperthyroid patients did not influence T4, T3U, FT4
index and TSH, but T3 decreased ( p < 0.001) as of day 2, and rT3 increased
(p<0.05). Plasma-T3 declined in all 11 patients, in four to within the
normal range (figure VII.2). The ratio's of rT3/T3 (p<0.01) and rT3/T4
(p < 0.05) increased, the ratio T 3 /T 4 decreased (p < 0.001). The TSH-
response oh TRH stimulation remained absent; T3-levels during the
second test were lower than during the first test (p < 0.001.)
In all hypothyroid patients on L-thyroxine substitution plasma-T3
decreased during propranolol medication, in two to subnormal values
(figure VII.2); T3-values were lower (p< 0.001) as of day 5. Reverse T 3 (p<
0.02) T4 (p < 0.001) and TSH (p < 0.001) increased, T3U did not change.
The ratio's of rT3/T3 (p < 0.01) and rT3/T4 (p< 0.02) increased, the ratio
T,/T4 decreased (p < 0.0\). The TSH-levels after TRH stimulation during
 ,, 7 W r^-.-~;.-ya^S3«£S^^

K//-2
TRH-stimulation test before propranolol and during 160 mg propranolol; TSH-values are presented as the geometric mean ± SD in mU/1, and
T3-values as x ± SD in nmol/1.

minutes 0 20 60 120 180 240

group A. hyperthyroid patients (n = 11)

TSH before propranolol <0.3 <0.3 <0.3 <0.3 <0.3 <0.3

TSH during 160 mg
propranolol <0.3 <0.3 <0.3 <0.3 <0.3 <0.3
T3 before propranolol 4.40.± 1.23 4.16 ± 1.23 4.33 ± 1.23 4.36 ± 1.23 4.84 ± 1.48 4.31 ± 1.39
T3 during 160 mg
propranolol 3.20 ± 1.16 3.34 ± 1.29 3.47 ± 1.17 3.54 ± 1.17 3.67 ± 1.20 3.68 ± 1.40

group B. hypothyroid patients ori L-T4 (n = 6)

TSH before propranolol 1.4 4.0 3.9 2.7 2.3 2.0

«0.3 -10) (0.3 -47) (0.3 - 47) (0.3 - 28) ( 0 . 3 - 20) K0.3 - 1 6 )
TSH during 160 mg 2.2 5.2 5.6 4.4 3.3 2.2
propranolol ( 0.3 -19) ( 0 . 3 - 86) (0.5 - 67) (0.4 - 46) ( 0 . 4 - 31) «0.3 -23)
T3 before propranolol 1.56 ± 0.39 1.59 ± 0.34 1.68 ± 0.46 1.65 ±0.51 1.72 + 0.32 1.77 ± 0.39
T 3 during 160 mg
propranolol 1.29 ± 0.42 1.31 + 0.49 1.26 ± 0.43 1.29 ± 0.34 1.40 ± 0.43 1.37 ± 0.35

!!!^?R?K^ - r — . - - . - - - •• • •••-• •-'-•" ••<•• '• "

 162
GROUP A - HYPERTHYROIDISM GROUP B- HYPOTHYROIDISM ON L-T« j
1

6.15 2.30

5.70 2.15

525 200
4.80 1.85
4.35 1.70
3.90 1.55
3.45 1.40
3.00 1.25
2.55 1.10

2.10 0.95
1.65 • 0.80 \

1.20 0.65

160 0 160
propranolol propranolol nig/day

Figure VII.2. Decrease in plasma T, during propranolol treatment in each individual

v
hyperthyroid and hypothyroid patient. ;

GROUP A - HYPERTHYROIDISM GROUP B- HYPOTHYROIDISM ON L-T«

'i- I
T3 230
nmol/, 5.05 200
430 1.70
3.55 1.40 " - .
260 1.10
2.05 0B0

7. • 10
10
change '
from 0 0
baseline
T3
-10
-20 -20
-30 -30
-40 -40
-50 -50

0 80 160 0 80 160
propranolol m g / d a y propranolol m g / d a y

Figure VII.3. Absolute and relative decrease in plasma T, during propranolol treatment in the
hyper- and hypothyroid patients (mean ± SD).
 163
propranolol medication were not higher in comparison with the
pretreatment levels (p > 0.1); T3-levels during this test were lower
i (p<0.05) on propranolol.
Figure VII.3 depicts the absolute and relative decrease in plasma-T3 of
groups A and B. The mean per cent change from pretreatment plasma-T3
in the hyperthyroid patients is -17% (range: +6% to -38%) after one week
and -31% (range: -16% to -55%) after the second week. In the hypothyroid
patients these values are -17% (range: -8% to -34%) and -28% (range: -13%
to -55%). Statistical analysis indicated no differences in per cent decrease %
between the hyper- and hypothyroid groups. The corresponding figures I
for the mean per cent change in plasma-rT3 are: group A, after one week
+20% (range: -23% to +85%) and +30% (range: -20% to+118%) after the
second week; group B, after one week +46% (range: -8% to +233%) and
+77% (range: +8% to +300%) after the second week.

VII.4. Discussion.

The results of this study indicate that propranolol treatment lowers

plasma-T3 in hyperthyroid patients and in hypothyroid patients on L-
thyroxine substitution therapy. The decrease in plasma-T3 cannot be
explained by an in vitro effect of propranolol on the T3-
radioimmunoassay. Propranolol even in a concentration of 250//g/ml
plasma did not influence the assays of T3 and TSH; it has been
demonstrated that plasma levels of propranolol during treatment with 160
mg per day are in the order of 0.1/*g/ml (Evans and Shand 1973; Niesand
Shand 1975). Interference of propranolol or its metabolites with plasma
protein binding sites for thyroid hormones is highly unlikely; the T3U ratio
remained constant, and plasma-T4 did not decrease concomitantly.
Furthermore, the rise in TSH-and T4-levels in the hyothyroid group points
very strongly to a real effect of propranolol on the secretion and/or
metabolism of thyroid hormones. However, impairment of T3-secretion by
the thyroid in the hyperthyroid patients seems not to be a major effect of
the drug (given in this dosage), since a decrease in plasma-T3 of the same
magnitude was found in the hypothyroid patients. Peripheral conversion
of exogenous T4 was the main, if not the only source of plasma T3 and rT3
in these patients, because any residual thyroid function was suppressed by
substitution therapy. The concomitant rise in plasma rT3 strongly suggests
impairment of the 5'-deiodination of T4 as the cause for the reciprocal
changes in the triiodothyronines. A similar decrease in plasma T3 and
increase in plasma rT3 concentrations has been found in subjects treated
with propylthiouracil (Abuid and Larsen 1974; Saberi et al 1975; Geffner
et al 1975; Westgren et al 1977; Siersbaek-Nielsen et al 1978; Laurbergand
Weeke 1978), dexamethasone (Chopra et al 1975; Burr et al 1976;
Westgren et al 1977), amiodarone (Burgeret al 1976; Jonckheer et al 1978)!
 -•r—j-

164
iopanoic acid (Biirgi et al 1976), and ipodate (Wu et al 1978). Kinetic
studies of Lumholtz et al (1978,1979) in hypothyroid patients maintained
on L-T4 replacement therapy have demonstrated a decrease in the T4-T3
conversion rate (but no change in the T4*rT3 conversion rate), and a
decrease in the MCR of rT3 during propranolol treatment. These findings
constitute conclusive evidence that propranolol inhibits the 5'-
deiodination of T4.
The reciprocal changes in plasma T3 and rT3 under propranolol
medication have been reported by many authors (Nauman et al 1974;
Murchison et al 1976; Harrower et al 1977; Lotti et al 1977; Theilade et al
1977; Verhoeven et al 1977; Wiersinga and Touber 1977; Kallner et al 1978;
Saunders et al 1978; Tevaarwerk et al 1978; Faber et al 1978; Feely et al
1979). In retrospect, Nauman et al were the first who described the
decrease in plasma T3, and Verhoeven et al first reported the increase in
plasma rT3. The mean per cent decrease in plasma T3 in these studies varies
between 16 and 40%, according to the dose and duration of propranolol
medication; the mean per cent increase in plasma rT3 is reported as 16-
43%. The decrease in plasma T3 is correlated to the plasma propranolol
concentration (Feely et al 1979). This may explain the variable responses
seen in individual patients, since plasma propranolol levels may vary
considerably (Rubenfeld et al 1979).

We observed an increase in T4 and TSH during propranolol medication

i
in the hypothyroid patients on L-T4 substitution therapy. A rise in TSH is
also reported by Feely et al (1979) and — although not significantly — by
Faber et al (1979); however, an increase in T4 is not observed in other
studies (Lumholtz et al 1978, 1979; Feely et al 1979; Faber et al 1979).
Similar changes in T4 and TSH are reported in L-T4 treated subjects during
administration of other drugs that inhibit the deiodination of T4:
1) Propylthiouracil medication results in an increase of TSH but not of
T4 levels in the studies of Saberi et al (1975) and Geffner et al (1975),
although no change in TSH (Siersbaek-Nielsen et al 1978) and an
increase in T4 (Westgren et al 1977) is also reported.
2) Treatment with amiodarone resulted in a slight but not significant rise
in T4; TSH, however, remained undetectable possibly due to the high
ii
substitution dose of L-T4 (300 /Ag) (Burger et al 1976).
3) Iopanoic acid medication increased TSH but was without effect on T4 i
(Bflrgi et al 1976).
4) Administration of ipodate increased T4, but did not alter TSH; how-
ever, in 2 of the 4 patients studied TSH was undetectable (Wu et al 1978).
5) The studies on dexamethasone are of no use in this respect, since
1
corticosteroids interfere with the release of TSH (Wilber and Utiger
1969; Nicoloff et al 1970; Sowers et al 1977).
These studies therefore indicate that inhibition of the 5'-deiodinationof T4
 165
r
is often associated with a rise in T4 and TSH.
With regard to our own results, we suggest that the increase in TSH is
caused by the fall in plasma T3 — a direct effect of propranolol on the
release of TSH could not be demonstrated in several studies (Woolf et al
1972; Epstein et al 1975; Wartofsky et al 1975; Lauridsen et al 1976)—and
that the increase in T4 is caused by a lower MCR of T4 due to the inhibition
if of the 5'-deiodination of T4. For obvious reasons an increased thyroidal
i release of T4 in the L-T4 treated subjects can be ruled out; moreover, a
direct effect of propranolol on thyroidal release was absent in a study of
Wartofsky et al (1975). The kinetic studies of Lumholtz et al (1978,1979) in
L-T4 treated hypothyroid patients on propranolol medication indeed
demonstrate a decreased MCR of T4; however, the MCR of T3 and rT3 are
also lowered by the drug, so that no clear conclusion can be drawn on the
contribution of the impaired deiodination to the decrease in MCR of T4.
Studies in euthyroid subjects treated with one of the above-mentioned
drugs further support the proposed explanation, although the picture is
complicated by the capability of the normal thyroid to respond to
increased TSH levels. Plasma T4 concentration increases during treatment
with amiodarone, iopanoic acid or ipodate, and these drugs also induce a
rise in plasma TSH (with the exception of ipodate).
Propranolol medication in euthyroid subjects also decreases plasma T3
and increases plasma rT3 concentrations (Kristensen et al 1978; Faber et al P
1979). Plasma TSH does, however, not change (Faber et al 1979), and i
plasma T4 either remains unaltered (Lotti et al 1977; Shenkman etal 1977;
Faber et al 1979) or increases (Williams and Jacobs 1970; Kristensen and
Weeke 1977). Again, chronic propranolol treatment in the intact or
hypophysectomized rat increases plasma T4 (Tal et al 1972).
More detailed studies in a greater number of subjects with more frequent
blood sampling may further elicit the effects of pharmacological inhibition
of 5'-deiodination on the plasma levels of T4 and of TSH. Nevertheless, the
rise in TSH and the rise in T4 (probably due to both TSH-stimulated
thyroidal release and to a decreased MCR) subsequent to impaired 5'-
deiodination of T4, as observed in a relatively large number of patients with
AMI (see chapter VI), strongly favours the mechanism suggested.

The nature of the inhibitory effect of propranolol on the 5'-deiodination

of T4 is unclear. Dratman (1974) has hypothesized that T4, an amino acid
analog, has a neurotransmitter function in the adrenergic nervous system,
and that T4 is deiodinated to T3 by tyrosine hydroxylase, the initial enzyme
in the synthesis of the catecholamines. However, administration of a-
methyl-p-tyrosine (a-MPT), an inhibitor of tyrosine hydroxylase, to L-T4
treated human subjects did not change the plasma T4 or T3 concentrations
(Dvorak etal 1978). Also, a -MPT failed to influence the T4 -T 3 conversion
in vivo or in vitro in animal studies (Chopra 1977; Hiifner et al 1977;
•!: 166 t
|i Kaplan a n d Utiger 1978; Pascual et al 1978). The hypothesis of Dratman . |;
?f' therefore seems unlikely. On the other hand, in-vitro studies on the effect |£
Fjj of propranolol (chapter VIII) indicate that intact{3-adrenergic receptors l|\
g are necessary for the inhibitory effect on the deiodination of T 4 . It is thus ff.
| conceivable, that changes in the adenylate-cyclase/ cyclic A M P system , ft
| secondary t o stimulation or blockade of thefl-adrenergic receptors play a %
g role in the modulation of the 5'-deiodinating activity. g
!
?;i The biological significance of the 'low T 3 syndrome' induced by |:
I propranolol is incompletely understood. The decrease in plasma T 3 may
|; explain in part the benificial effects of 0-adrenergic blockade in
: hyperthyroid patients: the per cent reduction of plasma T 3 is directly
' related to the fall in oxygen consumption (Saunders et al 1978) and to the
i degree of continued weight loss or weight gain (Feelyetal 1979). However,
:• 0 -adrenoceptor blocking agents d o not suppress all peripheral mani-
|. festations of thyroid hormone excess (McDevitt et al 1968; Grossman '.
]- et al 1971; Malcolm 1972; Zwillich et al 1978), and have many actions other '
[ than the S'-deiodination inhibition. The decrease in T 3 thus seems to be
[ only partially responsible for the clinical effects of propranolol in
jjj hyperthyroidism.
[I It has been suggested that the propranolol withdrawal syndrome,
\{ characterized by tachycardia, angina pectoris, sweating or tremor
\\ (resembling the signs and symptoms of thyrotoxicosis), is due to a rebound
'• increase of the suppressed plasma T3 (Kristensen et al 1978). However, it is
more likely that the withdrawal syndrome is causally related to the
increased number of 0-adrenergic receptors induced by propranolol
treatment (Glaubiger and Lefkowitz 1977), with a consequent transient
hypersensitivity to j3-agonists after sudden withdrawal of propranolol
(Boudoulas et al 1977; Nattel et al 1979). Naturally, an increase in plasma
T3 may well aggravate the hypersensitivity to (5-agonists.

I VII.5. References.

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| the acute changes during therapy with antithyroid agents. J Clin Invest 1974; 54: 201-8.
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§ York: John Wiley and Sons, 1954. ;
i Biran S, Tal E. Effect of beta-blocking drug propranolol on m I utilization in euthyroid
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J Boudoulas H, Lewis R P, Kates R E, Dalamangas G. Hypersensitivity to adrenergic stimula- :
I ; tion after propranolol withdrawal. Ann Int Med 1977; 87: 433-6. i
j Burger A, Dinichert D, Nicod P, Jenny M.Lemarchand-BeraudTh, Vallotton MB. Effect of I
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r amiodarone on serum triiodothyronine, reverse triiodothyronine, thyroxin, and | '
; thyrotropin. A drug influencing peripheral metabolism of thyroid hormones. J Clin •
| Invest 1976; 58:255-9. \
| Bflrgi H, Wimpfheimer C, Burger A, Zaunbauer W, Rdsler H, Lemarchand-Beraud Th. \
-?~, Changes of circulating thyroxine, triiodothyronine and reverse triiodothyronine after
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'$£' Effects of a single dose of dexamethasone on serum concentrations of thyroid hormones^
re Lancet 1976; 2: 58-61.
1} Chopra 1 J, Williams D E, Orgiazzi J, Solomon D H. Opposite effects of dexamethasone on
[ serum concentrations of 3,3',5'-triiodothyronine (reverse T,) and 3,3',5-triiodothyronine
fj (T3) in man. J Clin Endocrinol Metab 1975; 41: 911-20.
i| Chopra I J. A study of extrathyroidal conversion of thyroxine (T4) to 3,3',5-triiodothyronine*
(T3) in vitro. Endocrinology 1977; 101: 453-63.
Dratman M B. On the mechanism of action of thyroxine, an amino acid analogue of tyrosine.
J Theor Biol 1974; 46: 255-70.
Duncan D B. Multiple range and multiple F tests. Biometrics 1955; 11: I.
Dvorak J C, Engelman K, Utiger R D. Failure of a-methyltyrosine to inhibit peripheral
triiodothyronine formation. J Cltn Endocrinol Metab 1978; 47: 442-4.
Epstein S, Pimstone B L, Vinik A I, McLaren H, Failure of adrenergica and (3 receptor block-
ade to elevate the TSH and prolactin response to TRH in hyperthyroidism. Clin
Endocrinol 1975; 4: 501-4.
Evans G H, Shand D G. Disposition of propranolol. V. Drug accumulation and steady-state
concentrations during chronic oral administration in man. Clin Pharmacol Ther 1973;
14: 487-93.
Faber J, Friis T, Kirkegaard C et al. Serum T 4 , T 3 and reverse T3 during treatment with
propranolol in hyperthyroidism, L-T4 treated myxedema and in normal man. Horm
Metab Res 1979; 11:34-6.
Feely J, Isles T E, Ratcliffe W A, Crooks J. Propranolol, triiodothyronine, reverse triiodo-
thyronine and thyroid disease. Clin Endocrinol 1979; 10: 531-8.
Geffner D L, Azukizawa M, Hershman H M. Propylthiouracil blocks extrathyroidal conver-
sion of thyroxine to triiodothyronine and augments thyrotropin secretion in man. J Clin
Invest 1975; 55: 224-9.
Glaubiger G, Lefkowitz R J. Elevated beta-adrenergic receptor number after chronic propra-
nolol treatment. Biochem Biophys Research Commun 1977; 78: 720-5.
Grossman W, Robin N I, Johnson L W, Brooks K, Selenkow H A. Effects of beta-blockade
on the peripheral manifestations of thyrotoxicosis. Ann Int Med 1971; 74: 875-9.
Hadden D R, Montgomery D A D , Shanks R G, Weaver J A. Propranolol and iodine-131 in
the management of thyrotoxicosis. Lancet 1968; 2: 852-4.
Hadden D R, Bell T K, McDevitt D G, Shanks R G, Montgomery D A D , Weaver J A. Pro-
pronolol and the utilisation of radioiodine by the human thyroid gland. Acta Endocrinol
1969; 61: 393-9.
Harrower A D B, Fyffe J A, Horn D B, Strong J A. Thyroxine and triiodothyronine levels in
hyperthyroid patients during treatment with propranolol. Clin Endocrinol 1977; 7:41-4.
Hiifner M, Grussendorf M, Ntokalou M. Properties of the thyroxine (T4) monodeiodinating
system in rat liver homogenate. Clin Chim Acta 1977; 78: 251-9.
Jonckheer M H, Blockx P, Broeckaert I, Cornette C, Beckers C. 'Low T3 syndrome' in
patients chronically treated with an iodine-containing drug, amiodarone. Clin Endocrinol
1978; 9: 27-35.
Kallner G, Ljunggren J-G, Tryselius M. The effect of propranolol on serum levels of T 4 , T3
and reverse-Tj in hyperthyroidism. Acta Med Scand 1978; 204: 35-7.
Kaplan MM, Utiger RD. Iodothyronine metabolism in rat liver homogenates. J Clin Invest
> 1978; 61: 459-71.
j. Krikler D M. Adrenergic receptor blockade. Lancet 1966; 1: 268.
| Kristensen B 0 , Weeke J. Propranolol induced increments in total and free serum thyroxine in
I patients with essential hypertension. Clin Pharmacol Ther 1977; 22: 864-7.
 168
Krktensen B 0 , Steiness E, Weeke J. Propranolol withdrawal and thyroid hormones in
patients with essential hypertension. Clin Pharmacol Ther 1978; 23:624-9.
Landsberg L. Catecholamines and hyperthyroidism. Clin Endocrinol Metab 1977; 6:697-718 #!:-
Laurberg P, Weeke J. Opposite variations in serum T3 and reverse T3 during propylthiouracil
treatment of thyrotoxicosis. Acta Endocrinol 1978; 87: 88-94.
Lauridsen U B, Faber J, Friis Th, Kirkegaard C, Nerup J. Thyrotropin (TSH) release during
altered adrenergic a and j8 receptor influence. Horm Metab Res 1976; 8: 406-7.
Levey G S. Catecholamine sensitivity, thyroid hormone and the heart, a reevaluation. Am J
Med 1971; 50: 413-20.
Levey G S. The heart and hyperthyroidism: use of beta-adrenergic blocking drugs. Med Clin
N Am 1975; 59: 1193-1201.
r Lotti G, Delitala G, Devilla L, Alagna S, Masala A. Reduction of plasma triiodothyronine
(T,) induced by propranolol. Clin Endocrinol 1977; 6: 405-10.
Lumholtz IB, Siersbaek-Nielsen K, Faber J, Kirkegaard C, Friis Th. Effect of propranolol on
extrathyroidal metabolism of thyroxine and 3,3',5-triiodothyronine evaluated by non-
compartmental kinetics. J Clin Endocrinol Metab 1978; 47: 587-9.
Lumholtz IB, Busch-Serensen M, Faber J.Friis Th, Kirkegaard C, Siersbaek-Nielsen K. The
influence of propranolol on the extrathyroidal metabolism of 3,3',5'-triiodothyronine
(reverse T3). Acta Med Scand 1979; suppl 624: 31-4.
Malcolm J. Adrenergic beta receptor inhibition and hyperthyroidism. Acta Cardiol 1972; 27,
suppl XV: 307-26.
McDevitt D G, Shanks R G, Hadden D R, Montgomery D A D , Weaver J A. The role of the
thyroid in the control of heart-rate. Lancet 1968; 1: 998-1000.
McLarty D G,.Brownlie B E W, Alexander W D, Papapetrou P D, Horton P. Remission of
thyrotoxicosis during treatment with propranolol. Brit Med J 1973; 2: 332-4.
Murchison L E, Bewsher P D, Chesters M I, Ferrier W R. Comparison of propranolol and
practolol in the management of hyperthyroidism. Brit J Clin Pharmacol 1976; 3:273-7.
Nattel S, Rangno R E, Loon G van. Mechanism of propranolol withdrawal phenomena.
Circulation 1979; 59: 1158-64.
Nauman J, Nauman A, Roszkowska K. Influence of propranolol on levels of thyroxine and
triiodothyronine in hyperthyreotic patients. Mat Med Pol 1974; 6: 178-82.
Nicoloff J T, Fisher D A, Appleman M D. The role of glucocorticoids in the regulation of
thyroid function in man. J Clin Invest 1970; 49: 1922-9.
Nies A S, Shand D G. Clinical pharmacology of propranolol. Circulation 1975; 52: 6-15.
Pascual A, Obregon M J, Morreale de Escobar G. Tyrosine hydroxylase and the conversion
of L-thyroxine into 3',3,5-triiodo-L-thyronine in the rat. Endocrinology 1979; 104:1574-
9.
Ramsay I. Adrenergic j3-receptor blockade in hyperthyroidism. Brit J C!in Pharmacol 1975;
2: 385-8.
Rubenfeld S, Silverman V E, Welch K M A, Mallette L E, Kohler P O. Variable plasma
propranolol levels in thyrotoxicosis. New Engl J Med 1979; 300: 353-4.
Saberi M, Sterling F H, Utiger R D. Reduction in extrathyroidal triiodothyronine produc-
tion by propylthiouracil in man. J Clin Invest 1975; 55: 218-23.
Saunders J, Hall S E H, Crowther A, SSnksen P H. The effect of propranolol on thyroid
hormones and oxygen consumption in thyrotoxicosis. Clin Endocrinol 1978; 9: 67-72.
Shanks R G, Hadden D R, Lowe D C, McDevitt D G, Montgomery D A D . Controlled trial
of propranolol in thyrotoxicosis. Lancet 1969; 1: 993-4.
Shenkman L, Podrid P, Lowenstein J, Hyperthyroidism after propranolol withdrawal.
JAMA 1977; 238: 237-9.
Siersbaek-Nielsen K, Kirkegaard C, Rogowski P, Faber J, Lumholtz B, Friis Th. Extra-
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TRH-induced TSH-release in man. Acta Endocrinol 1978; 87: 80-7.
 .-M

169 f^
Sowers J R, Carlson H E, Brantbar N, Hershman J N. Effect of dexamethasone on prolactin •£•;
and TSH responses to TRH and metoclopramide in man. J Clin Endocrinol Metab 1977; «'i
44:237-41. @
Spaulding S W, Noth R H. Thyroid-catecholamine interactions Med Clin N Am 1975; 59: %
1123-31. S:
TalE, BiranS, Sulman F G. Influence of propranolol on serum thyroxine in the rat. JEndo- ? %\
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Tevaarwerk G J M , Malik M H, Boyd D. Preliminary report on the use of propranolol in ¡v
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Theilade P, Hansen J M, Skovsted L et al. Propranolol influences serum T3 and reverse T3 in !
hyperthyroidism. Lancet 1977; 2:363. : ¡if
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thyroid iodine release, thyroxine turnover, or TSH and PRL response to thyrotropin- : ;
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485-90. • j |
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 'I

CHAPTER VIII.

STUDIES ON THE EFFECT OF PROPRANOLOL IN ISOLATED

RAT LIVER PARENCHYMAL CELLS

VIII. 1. Introduction.

In man several drugs have been shown to influence the peripheral

conversion of T4. Propylthiouracil (Abuid and Larsen 1974; Saberi et al
1975; Geffner et al 1975; Westgren et al 1977; Siersbaek-Nielsenetal 1978;
Laurberg and Weeke 1978), dexamethasone (Chopra et al 1975; Burr et al
1976; Westgren et al 1977), amiodarone (Burger et al 1976; Jonckheer etal
1978), iopanoic acid (Biirgi et al 1976), and ipodate (Wu et al 1978), all
decrease plasma levels of T3 with a concomitant rise in plasma rT3 due to
inhibition of the 5'-deiodination process. These drugs also inhibit the T4-T3
(and when investigated the rT3-»3,3'-T2) conversion in vitro as studied in rat
liver or kidney cell preparations; the only exception are the corticosteroids,
which do not inhibit the conversion of T4 when added directly in vitro, but
decrease the conversion after pre-treatment of the animal in vivo (Visser et al
1975; Hiifner and Kndpfle 1976; Chopra 1977; Balsam et al 1978;
Chiraseveenuprapund et al 1978; Chopra et al 1978; Heyma et al 1978;
HOffken et al 1978; Htifner and Grussendorf 1978; Kaplan and Utiger 1978;
Leonard and Rosenberg 1978; Visser et al 1978).
We have previously demonstrated that the B-adrenoceptor blocking
agent propranolol lowers the plasma level of T3 in hyperthyroid patients and
hypothyroid patients on L-thyroxine substitution therapy (Wiersinga and
Touber 1977). Again, inhibition of the peripheral conversion seemed the
most likely explanation for this finding. Since the liver is a major site of T4-
deiodination the decrease in plasma T3 - and presumably, in T3 production -
could be caused by a direct effect of propranolol on the parenchymal liver
cells. However, a decrease in T3 production as a consequence of a decrease in
liver perfusion caused by propranolol (Nies and Shand 1975) seemed
equally possible. In order to further elucidate the action of propranolol, we
studied the effect of the drug on liver cells in vitro. The isolated liver cell
preparation - rather than liver cell homogenate - was chosen because the
action of propranolol is mediated via blockade of the 13-adrenergic receptors
of the liver cell plasma membrane (Wolfe et al 1976).
 171
VIII. 2. Materials and methods.

Liver cells of two groups of male Wistar rats were used; mean weight ± SD
are215± 11 gin group A (n=4) and 298 ± 18gingroupB(n=12). The rats
were anaesthetized with nembutal and received intravenously 0.2 ml
heparin (1000 I.U.) dissolved in 0.8 ml 1% NaNO2 to prevent blood
coagulation and vascular contraction. Parenchymal liver cells were isolated
by perfusion of the liver with calcium-free phosphate buffered Ringer-
solution saturated with carbogen (pH 7.4, 37°C) containing 20 g/1 bovine
serum albumin and 0.6 g/1 collagenase (Sigma) (Berry and Friend 1969;
James 1977). The isolated cells were suspended in 3 ml of a Krebs-Ringer-
bicarbonate buffer (pH 7.4,37°C, containing 2.5 g/1 bovine serum albumin
but no collagenase) at a concentration of 10-20 mg protein per ml and
incubated at 37°C in slowly rotating tubes. Cell suspensions of each
individual rat of group A were incubated in duplicate with 1 .3,MM sodium-
L-thyroxine (Sigma) in the presence or absence of 50/iM carbimazole, 50
//M 6-propyl-2-thiouracil or 580/*M D,L-propranolol (I.C.I.). In the same
manner, cells of the rats of group B were incubated with 1.3//M sodium L-
thyroxine with or without D,L-propranolol at concentrations of 290,580
and 1 1 6 0 / J M respectively.
T 4 and the various drugs were added to the incubation mixture and 500/vl
samples were taken from the incubation mixture at times 0,15, 30 and 60
minutes. The samples were immediately transferred to 1 ml ice-cold
absolute ethanol. After centrifugation (2000 g, 15 min, 4°C) the clear
supernatants were stored at -20°C. As controls, liver cells were incubated in
the absence of T4, and T 4 was incubated in the absence of liver cells. The
protein concentration of the incubation mixture was determined by the
biuret method (Itzhaki and Gill 1964); the concentration served as a measure
of the number of cells present.
The T3 and T4 concentrations of the ethanol extracts (diluted if necessary)
were measured in duplicate by specific radioimmunoassays (Visser et al
1975; Chopra 1977), using 300 g/1 polyethyleneglycol (Cheung and
Haunwhite Jr. 1976) for the separation of the bound and free hormone
fractions. Samples presenting the standard curve were incubated under
identical conditions, i.e. with a final ethanol concentration of 6.7% (v/v).
The detection limit of the T3-assay is 0.1 nM, of the T4-assay 3nM. Cross-
reactivity of T4 in the T3-assay is< 0.1%; cross-reactivity of T3 in the T4-assay
is 0.3%. The coefficient of intra-assay variation of the T3-assay is 13.5% at a
level of 1.1 nM, 4.5% at 8.2 nM and 6.9% at 21.7 nM. Carbimazole,
propylthiouracil or propranolol in concentrations used in these
experiments, did not interfere in the T3- and T4-assays. Corrections were
made for base-line T3 hormone levels and for the losses of T3 and T 4 in the
ethanol extraction procedure (the recovery of the thyroid hormones is 76.0
± 4.5% (x ± SD). The amount of T3 generated by the liver cells from the
 172
added T4 was expressed as the conversion ratio, i.e. (nmol T3 formed/ nmol
T4 added) x 100%.
Statistical evaluation was performed by two ways analysis of variance
with replications, mixed model. The factors are rats and treatments and the
observations constitute slopes of regression lines. Each slope is determined
by a least square fit to the conversion ratio versus time.

VIII3. Results.

The endogenous T4 concentration of the liver cells (i.e. cells incubated in

the absence of T4) was below the detection limit of the T4-assay. The T3
concentration of the incubation mixtures varied between 0.1 and 1.0 nM in
the absence of T4, and between 5 and 10 nM immediately afterthe addition
of 1300 nM T4. The increase in base-line T3 in the presence of T4 is due to
contamination with T3 « 0.5%) of the T4 preparation used and to the
immunological cross-reactivity of T4 in the T3-assay. T3 generation could

I 1
s

100 l 4 nnwl'l 200 60 90 120

ISO T,nmol/I TIME IminulM)
0.75

Figure VIII. 1. Demonstration of similarity between standard curves (•) and dilutions of
ethanol extracts (•) from isolated rat liver cells in the radioimmunoassays of T3 and T4.

Figure VIII.2. Conversion of exogenous T4 (1.3.//M) into T3 by isolated rat liver cells (protein
content 7.9 mg/ml).
 . 173
Table VIII-1
"$••

Effect of carbimazole, propylthiouracil and propranolol on the conversion of exogenous

T4 (1.3 MM) into T 3 by isolated rat liver cells. Conversion ratios are given as the mean W
± SEM (group A, n = 4; mean protein content ± SD is 18.9 ± 3.8 mg/ml).

Incubation time conversion ratio (nmol T 3 formed/nmol T, added x 100%)

in minutes controls carbimazole PTU propranolol
50 nM SOjuM 580/iM

IS 0.49 ± 0.10 0.39 ± 0.13 - 0 . 0 5 ± 0.02 0.16 ± 0.03

30 0.86 ± 0.20 0.78 ± 0.18 - 0 . 0 3 ± 0.06 0.21 ± 0.10
60 1.06 ± 0.25 1.10 ± 0.06 - 0 . 0 3 ± 0.06 0.26 ± 0.10

not be detected in liver cells incubated in the absence of T4, nor in T4

incubated in the absence of liver cells.
Measurements of T4 and T3 in diluted ethanol extracts resulted in dose
response curves similar to the standard curves (fig. VIII. 1). Figure VIII.2
depicts an example of T3 generation from exogenous T4 by isolated rat liver
cells; in general approximately 10 nM T3 is formed after 30 min. incubation.

o
I1 0 \!
i

o.s

TIME (mlnulsi) TIME (minules)

Figure VIII.3. Conversion of exogenous T4 (1.3//M) into T5 by isolated rat liver cells (group
A, n = 4; mean protein content ± SD is 18.9 ± 3.8 mg/ml). Conversion ratios are given as
mean ± SEM. • • controls, * — * carbimazole 50//m,o o proprylthiouracil 50//M,
• • propranolol

Figure VIII.4. Conversion of exogenous T4 (1.3.^M) into T3 by isolated rat liver cells (group
B, n = 12; mean protein content ± SD is 18.1 ± 1.1 mg/ml). Conversion ratios are given as
mean ± SEM. • • controls,* * propranolol 290 pM. • • propranolol 580//M,
o o propranolol 1160//M.
 .--J,—-

174
Table VUI-2
Effect of various concentrations of propranolol on the conversion of exogenous T4
(1.3 MM) into T3 by isolated rat liver cells. Conversion ratios are given as the mean ± SEM
(group B, n = 12; mean protein content ± SD is 18.1 + 1.1 mg/ml).

Incubation time conversion ratio (nmol T 3 formed/nmol T4 added x 100%)

in minutes controls propranolol propranolol propranolol
t 290 MM 580 juM 1160 MM

15 0,52 * 0.06 0.34 ± 0.05 0.21 ± 0.02 0.26 ± 0.05

30 0.82 ±0.11 0.69 ±0.12 0.55 ± 0.10 0.56 ± 0.09
60 1.47 ± 0.20 1.46 ±0.16 1.11 ±0.13 0.99 ± 0.16

The results of the experiments with the cells of rats of group A are
presented in table VIII. 1 and figure VIII. 3. In the statistical analysis
indications were found for the existence of interactions and treatments (p <
0.01). We used linear contrast (Armitage 1971) for the evaluation between ft
control and treatments. No difference in conversion ratios was found
between control and 50 pM carbimazole (p> 0.1). However, both 50//M
propylthiouracil and 580 pM propranolol lowered conversion ratios (p<
0.01). No interactions are found in the experiments of group B (results are
given in table VIII.2 and figure VIII.4). The interactions- and residual sum
of squares were pooled. The analysis was continued by a stepwise multiple
comparison procedure for ordered parameters (Spjotvoll 1977). The level of
significance was taken as a = 0.05. Addition of 580 and 1160//M
propranolol both resulted in lower conversion ratios, but no difference was
found between the 580 and 1160 juM dose. No inhibitory effect on the
conversion was found for 290/*M propranolol.

V1II.4. Discussion.

Our results indicate that isolated rat liver cells are suitable for the in vitro
study of the conversion of T4 into T3; T3 generation cannot be detected in the
absence of T4 nor is there a change in T3 level when T4 is incubated without
cells. We have applied conversion ratios because the deiodinating capacity
of the liver cells is not saturated at the T4 concentrations used (Hesch et al
1975; Chopra 1977).
Conversion of T4 into T3 by isolated liver cells has been reported
previously by Hesch et al (1975). Using a comparable amount of cells, they
found a lower conversion ratio (0.1%) than in the present study (0.8%). Rat
liver homogenate has also been shown to generate T3 from T4 (Visser et al
1975; HuTner and Knfipfle 1976; Visseretal 1976; Chopra 1977;Hiifneretal
1977); conversion ratios in this model were somewhat higher dependent on
the concentrations of substrate and (cell) protein. The results of the

•nrr-y
 I

175
experiments with the cells of the rats of group A show quite clearly that
whereas the addition of carbimazole has no effect on the conversion process,
propylthiouracil inhibits T,-generation. This is in accordance with the
results of studies using rat liver homogenate (Visser et al 1975; Chopra
1977).
An effect of propranolol on the in vitro conversion of T4 has thus far not
been reported. Our data show that this j}-adrenoceptorblockingagentalso
inhibits the conversion of T4 into T3 by isolated rat liver cells. The effect of
propranolol is not an artefact, since the drug did not influence either the T3
levels of cells incubated without T4 or the radioimmunoassays of T3 and T4. I
Chiraseveenuprapund et al (1978) could not demonstrate an effect of
propranolol (added in a dose of 2000/4M) on the T4 -»T3 conversion in rat
kidney homogenate; the discrepancy with our results is most likely
explained by the destruction of the B-adrenergic receptors in their
homogenization procedure.
We have been unable to establish an unequivocal dose dependent effect of
propranolol. Although both 580 and 1160 y/M propranolol inhibit the
conversion process while 290juM d oes not, there is no difference between the
effects of 580 and 1160 /*M.

Since propranolol is rapidly metabolised by the liver (Nies and Shand

1975) low levels of propranolol may not be sufficient to initiate or to I
maintain an inhibitory effect. This possibility might be explored in
experiments using 0-adrenoceptor blocking agents which are not
metabolised by the liver, such as atenolol and practolol.
The concentrations of propranolol used in our incubation mixture may
have been much higher than in vivo portal plasma levels of subjects given the
usual therapeutic dosage. The results of the studies of Hayes and Cooper
(1971) indicate that, in the dog, propranolol concentrations in liver tissue
are in the order of 0.5//g/ g (about ten times higher than the corresponding
blood levels) after the administration of 1 mg/kg orally. It is therefore
conceivable that in our experiments other than the (3-blocking properties of
the drug - such as the membrane stabilising activity - may have influenced
the results. Studies with B-blocking agents which lack this activity (e.g.
atenolol and metoprolol) are required to further elucidate the mechanism of
action in this respect.
In the rats of group A propranolol seems to inhibit T4 conversion more
effectively than in the rats of group B. This difference may well be due to the
difference in age: the rats of group B are older than those of group A.
In conjunction with our earlier finding (Wiersinga and Touber 1977) of
the in vivo lowering of plasma levels of T3 by propranolol, the
demonstration of the in vitro inhibitory effect of this drug underlines the
I
importance of B-adrenoceptor agonists and antagonists in the metabolism
and possibly ultimate biological effect of thyroid hormones.
 176
VIII. 5. References.

Abuid J, Larsen PR. Triiodothyronine and thyroxine in hyperthyroidism. Comparison of

the acute changes during therapy with antithyroid agents. J Clin Invest 1974; 54:201-8.
Armitage P. Statistical methods in medical research. Blackwell Scientific publications,
Oxford 1971.
Balsam A, Ingbar SH. The influence of fasting, diabetes, and several pharmacological
agents on the pathways of thyroxine metabolism in rat liver. J Clin Invest 1978; 62:415-24.
Berry MN, Friend DS. High-yield preparation of isolated rat liver parenchymal cells. A
biochemical and fine structural study. J Cellul Biol 1969; 43:506-20.
Burger A, Dinichert D, Nicod P, Jenny M, Lemarchand-Beraud Th, Vallotton MB. Effect of
amiodarone on serum triiodothyronine, reverse triiodothyronine, thyroxin, and thyro-
tropin. A drug influencing peripheral metabolism of thyroid hormones. J Clin Invest 1976;
58:255-9.
Btirgi H, Wimpfheimer C, Burger A, Zaunbauer W, R6sler H, Lemarchand-Beraud Th.
Changes of circulating thyroxine, triiodothyronine and reverse triiodothyronine after
radiographic contrast agents. J Clin Endocrinoi Metab 1976; 43:1203-10.
Burr WA, Ramsden DB, Griffiths RS, Black EG, Hoffenberg R, Meinhold H, Wenzel KW.
Effect of a single dose of dexamethasone on serum concentrations of thyroid hormones.
Lancet 1976; 2:58-61.
Cheung MC, Haunwhite Jr WR. Use of polyethylene glycol in separating bound from un-
bound ligand in radioimmunoassay of thyroxine. Clin Chem 1976; 22:299-304.
Chiraseveenuprapund P, Buergi U, Goswatni A, Rosenberg JN. Conversion of L-thyroxine
to triiodothyronine in rat kidney homogenate. Endocrinology 1978; 102:612-22.
Chopra IJ, Williams DE, Orgiazzi J, Solomon DH. Opposite effects of dexamethasone on
serum concentrations of 3,3',5'-triiodothyronine (reverse T,) and 3,3',5-triiodothyronine
(T3) in man. J Clin Endocrinoi Metab 1975; 41:911-20.
Chopra IJ. A study of extrathyroidal conversion of thyroxine (T4) to 3,3',5-triiodothyronine
(T3) in vitro. Endocrinology 1977; 101:453-63.
Chopra IJ, Wu S-Y, Nakamura Y, Solomon DH. Monodeiodination of 3,5,3'-triiodo-
thyronine and 3,3',5'-triiodothyronine to 3,3'-diiodothyronine in vitro. Endocrinology
1978; 102:1099-1106.
Geffner DL, Azukizawa M, Hershman HM. Propylthiouracil blocks extrathyroidal con-
version of thyroxine to triiodothyronine and augments thyrotropin secretion in man. J
Clin Invest 1975; 55:224-9.
Hayes A, Cooper RG. Studies on the absorption, distribution and excretion of propranolol in
rat, dog and monkey. J Pharmacol ExpTher 1971; 176:302-11.
Heyma P, Larkins RG; Stockigt JR, Campbell DG. The formation of tri-iodothyronine and
reverse tri-iodothyronine from thyroxine in isolated rat renal tubules. Clin Sci Mol Med
1978; 55:567-72.
Hesch RD, Brunner G, SOling HD. Conversion of thyroxine (T4) and triiodothyronine (T})
and the subcellular localisation of the converting enzyme. Clin Chim Acta 1975; 59:
209-13.
Hoffken B, Kodding R, Muhlen A von zur, Hehrmann T, Juppner H, Hesch RD. Regulation
of thyroid hormone metabolism in rat liver. Biochim Biophys Acta 1978; 539:114-24.
Httfner M, Knopfle M. Pharmacological influences on T4 to T, conversion in rat liver. Clin
Chim Acta 1976; 72:337-41.
Hiifner M, Grussendorf M, Ntokalou M, Properties of the thyroxine (T4) monodeiodinating
system in rat liver homogenate. Clin Chim Acta 1977; 78:251-9.
Hiifner M, Grussendorf M. Investigations on the deiodination of thyroxine (T4) to 3,3'-
diiodothyronine (3,3'-T2) in rat liver homogenate. Clin Chim Acta 1978; 85:243-51.
 177
L
-j ltzhaki RF, Gill DM. A micro-biuret method for estimating proteins. Anal Biochem 1964;
fl 9:401-10.
'd. James J. The genesis of polypoidy in rat liver parenchymal cells. Cytobiology 1977; 15:
» 410-9.
:?- Jonckheer MH, Blockx P, Broeckaert I, Cornette C, Beckers C. 'Low T3 syndrome' in
•: patients chronically treated with an iodine-containing drug, amiodarone. Clin Endocrinol •'.a

I: 1978:9:27-35.
I? Kaplan MM, Utiger RD. Iodothyronine metabolism in rat liver homogenates. J Clin Invest
% 1978; 61:459-71. '•3
|.; Laurberg P, Weeke J. Opposite variations in serum T3 and reverse T3 during propylthiouracil
|: treatment of thyrotoxicosis. Acta Endocrinol 1978; 87:88-94.
? Leonard JL, Rosenberg IN. Thyroxine 5'-deiodinase activity of rat kidney: observations
I on activation by thiols and inhibition by propylthiouracil. Endocrinology 1978; 103:
I 2137-44.
i Nies AS, Shand DG. Clinical pharmacology of propranolol. Circulation 1975; 52; 6-15.
; Saberi M, Sterling FH, Utiger RD. Reduction in extrathyroidal triiodothyronine production
i by propylthiouracil in man. J Clin Invest 1975; 55:218-23.
<. Siersbaek-Nielsen K. Kirkegaard C, Rogowski P, Faber J, Lumholtz B, Friis Th. Extra-
thyroidal effects of propylthiouracil and carbimazole on serum T4, T3, reverse T 3 and
TRH-induced TSH-release in man. Acta Endocrinol 1978; 87:80-7. 1,
Spjotvoll E. Ordering ordered parameters. Biometrica 1977; 64:327-34.
Visser TJ, van der Does-Tobe I, Docter R, Hennemann G. Conversion of thyroxine into
tri-iodothyronine by rat liver homogenate. Biochem J 1975; 150:489-93.
Visser TJ, van der Does-Tobe I, Docter R, Hennemann G. Subcetlular localization of a rat
liver enzyme converting thyroxine into tri-iodothyronine and possible involvement of
essential thiol groups. Biochem J 1976; 157:479-82.
Visser TJ, Fekkes D, Docter R, Hennemann G. Sequential deiodination of thyroxine in rat
liver homogenate. Biochem J 1978; 174:221-9.
Westgren U, Melander A, Wahlin E, Lindgren J. Divergent effects of 6-propylthiouracil on
3,5,3'-triiodothyronine (T3) and 3,3',5'-triiodothyronine (rT3) serum levels in man. Acta
Endocrinol 1977; 85: 345-50.
Westgren U, Ahren B, Burger A, Ingemansson S, Melander A. Effects of dexamathasone,
desoxycorticos^erone, and ACTH on serum concentrations of thyroxine, 3,5,3'-triiodo-
thyronine and 3,3',5'-triiodothyronine. Acta Med Scand 1977; 202:89-92.
Wiersinga WM, Touber JL. The influence of B-adrenoceptor blocking agents on plasma
thyroxine and triiodothyronine. J Clin Endocrinol Metab 1977; 45:293-8.
Wolfe BB, Harden TK, Molinoff PB. B-adrenergic receptors in rat liver: effects of adrena-
lectomy. Proc Nat Acad Sci USA 1976; 73:1343-7.
Wu S-Y, Chopra IJ, Solomon DH, Bennett LR. Changes in circulating iodothyronincs in
euthyroid and hyperthyroid subjects given ipodate (Oragrafin), an agent for oral
cholecystography. J Clin Endocrinol Metab 1978; 46:691-7.

:€>
I
CHAPTER IX.

GENERAL DISCUSSION
g
IX. 1. Physiological modulation of the peripheral conversion of T4.

The effect of physiological factors as sex, age and environment on the

conversion of T4 have been discussed in chapter III.4a. In man, sex- or
season-related differences have not been reported, but in the female rat the
T4~T3 conversion is lower than in the male, due to a decrease in the
concentration of the 5'-deiodinating activity.
The effect of age in man is debated; the frequently observed trend
towards lower plasma T3 and higher plasma rT3 concentrations in elderly
subjects (whether or not caused by co-existent non-thyroidal disease) is, I
however, almost certainly due to inhibition of the S'-deiodination process
(Wenzel and Horn 1976). More unanimity exists on the fate of the
iodothyronines in the fetal and neonatal period. There is no doubt that the
low T3 and high rT3 levels in the fetus are caused by inhibition of the S'-
deiodination of T4 (Harris et al 1978; see also chapter III.4c). The
preferential pathway of monodeiodination of T4 to rT3 has also been
demonstrated in the human fetus (Lightner et al 1977; Klein et al 1978).
Interestingly, the S'-deiodinating pathway is activated soon after birth
resulting in a rise of T3 and a afall of rT3 plasma levels (Chopra et al 1975) and
in fact, the increase in T4-»T3 conversion already begins some days before
labor in association with a rise in plasma cortisol, as demonstrated in the
sheep and the rat (Klein et al 1978; Wu et al 1978). With regard to the
mechanism of the 5'-deiodination inhibition during the fetal and neonatal
period, conflicting data have been published. Chopra (1978) reported a
marked dithiothreitol (DDT)-induced increase in the T4-»T3 conversion by
fetal sheep liver homogenates, suggesting that the low conversion is related
more to the availability of sulfhydryl groups than to a deficiency of the S'-
deiodinating enzyme. In contrast, Harris et al (1979) concluded that the
decreased T3 generation in the fetal rat is due to a decrease in the enzyme 1
concentration per se, while in the neonatal rat it is secondary to both a I
reduced availability of sulfhydryl groups and to a deficiency of the
deiodinating enzyme. Finally, Woeber (1978) found no difference in T3-
I
and rT3-generating activities between human polymorphonuclear
leucocytes taken from normal adults and from cord blood. Differences in
technical procedures and in species may explain some of the discrepancies.
 -r-vr-J-

179
';;). The nutritional state of the body is another prominent factor in the
?1 peripheral conversion of T4. It is appreciated that body weight is one of the
t: determinants of the plasma T3 concentration, high levels being found in
£ obesity (Bray et al 1976; Danforth 1978) and low levels—associated with a
!> high plasma rT3 — in malnutrition or simple weight loss (Chopra and •
'p Smith 1975; Ingenbleek and Beckers 1975; Vigersky et al 1977; Thomson et
li al 1977) and in anorexia nervosa (Miyai et al 1975; Moshang et al 1975;
§ Bradlow et al 1976; Boyar et al 1977; Croxson et al 1977; Leslie et al 1978). j
If Experimental obesity is associated with an increase of plasma T3 and
Fl sometimes with a decrease of plasma rT3 concentrations; the PR of T3 is
y increased in the overfed state, irrespective of the dietary composition, but
l{ T4 kinetics do not change (Danforth 1978). In contrast, starvation results
in a fall of plasma T3 and in a rise of plasma rT3 concentrations (Portnay et
: al 1974; Vagenakis et al 1975; Merimee and Fineberg 1976; Carlson et al \
i 1977; Croxson etal 1977; Palmblad etal 1977; Azizi 1978; Visseretal 1978;
I Burman et al 1979). Kinetic studies in fasting human subjects have i
[, demonstrated a decrease in the PR ofT3 and in the MCR of rT3, but also an -t,
| increased PR of rT3, indicating a lower T4^T3 and a higher T4*rT3
i conversion rate (Vagenakis et al 1977; Eisenstein et al 1978; Suda et al
\. 1978). The impairment of the 5'-deiodination of T4 has been confirmed in
'1 in vitro studies using liver homogenates from fasting animals (Balsam and
| Ingbar 1979; Gavin etal 1979; Harris etal 1979; Kaplan 1979). The nature ':
h of the defect in the 5'-deiodination process during fasting seems to be a
i' decrease in the concentration of sulfhydry 1 groups (leading to less reduced
v glutathione, the possible co-factor of the putative 5'-deiodinase); however,
according to Gavin et al (1979) and Kaplan (1979) a change in the enzyme
concentration is also involved.
The above-mentioned studies raise the possibility that some specific
dietary constituent rather than the total amount of ingested calories might
be responsible for the observed changes in the conversion of T4. Several
studies suggest that carbohydrates (CHO) are of prime importance in this
respect. Refeeding fasting human subjects with glucose results in a return
to normal of the plasma T3 and rT3 levels; refeeding with protein is without
I effect (Croxson et al 1977; Westgren et al 1977; Azizi 1978; Burman et al
| 1979). However, Davidson and Chopra (1979) recently suggested that the
I effect of non-CHO calories on the 5'-deiodination of T4 is more
f pronounced, if at least a normal amount of CHO (~200 g) is ingested. CKO
:
l and proteins, but not lipids, are modulators of the 5'-deiodination of T4 as *
? studied in rat liver homogenates (Harris et al 1978).

IX.2. Pathophysiological modulation of the peripheral conversion of T 4 . $

L; The effect of thyroidal disease upon the peripheral conversion of T4 in \

l man cannot be properly evaluated at the present time. The overproduction
 180
of T3 relative to T4 in hyperthyroidism is most likely caused by both
preferential secretion of T3 and increased T4-T3 conversion, since the
conversion rate of T4 to T3 in untreated hyperthyroid patients is higher
than after treatment. This increased conversion may well be induced by a
higher concentration of the substrate, T4. The conversion rate of T4 to T3 is
also higher in the untreated hypothyroid patient, despite the lower sub-
strate concentration. It is therefore speculated that the extrathyroidal
tissues have the capability to modulate the conversion of T4 in such a way
that, relative to rT3, less T3 is produced in hyperthyroidism but more in
hypothyroidism (cf. the ratio's of plasma iodothyronine concentrations
found in patients with thyroid function disorders, chapter IV). No data
are available on the generation of rT3 from T4 in states of thyroid hormone
excess or deficiency, neither in man nor in animals. In vitro studies with
liver and kidney homogenates from the rat and the dwarf mouse, have
shown that the 5'-deiodinating activity is stimulated in hyper-
thyroidism, and is inhibited in hypothyroidism: the production
of T3 from T4 and the degradation of rT3 are enhanced in
homogenates from hyperthyroid animals but inhibited in hypothyroid
animals (Grussendorf and Hufner 1977; Balsam et al 1978; Kaplan and
Utiger 1978; Harris et al 1979). This modulation of the peripheral
conversion of T4 is most likely due to changes in the concentration of the 5'-
deiodinase since the 5'-deiodination process is normalized after pre-
treatment of the hypothyroid animal with thyroid hormones, but not after
the in vitro addition of DTT (Harris et al 1979). It thus appears that T4 and
T3 serve as enzyme inductors. These in vitro observations do not
necessarily negate the speculation mentioned earlier: in this respect the
ratio's of the T4-*T3 and T4*rT3 conversion rates are more important than
the absolute values of the conversion rates.
Many non-thyroidal diseases exert a profound effect on the peripheral
conversion of T4. One wonders if not all non-thyroidal diseases, traumas,
surgical procedures etc. are associated with this phenomenon. As
discussed in chapter V, kinetic studies in patients with non-thyroidal
disease indicate an inhibition of the 5'-deiodination of T4; the low plasma
T3 is thus caused by a decreased conversion of T4 to T3, and the high plasma
rT3 mainly by a decreased metabolic clearance rate of rT3.

IX.3. Pharmacological modulation of the peripheral conversion of T4.

Several pharmacological agents widely used in clinical medicine inhibit

the S'-deiodination of T4 and are listed in table IX. 1. However,
carbimazole (Abuid and Larsen 1974; Saberi et al 1975; Laurberg and
Weeke 1978; Siersbaek-Nielsen et al 1978), lithium (Burman et al 1976;
Blomqvist et al 1977; Chiraseveenuprapund et al 1978) and iodide (Burger
et al 1976; Chiraseveenuprapund et al 1978) do not inhibit the peripheral

.-_,.
V conversion of T4. Conflicting reports have been published on the effect of
& diphenylhydantoin (Cullen et al 1973; Hflfnerand Knopfle 1976; Tsukuiet
•$ al 1978), and the influence of sex steroids depends apparently on the
I' experimental circumstances (Chen and Walfish 1978; Harris et al 1979) (cf.
i chapter III.4a). Polychlorinated biphenyls, which are widespread
I environmental pollutants, enhance the S'-deiodination process in animals
| (Bastomsky et al 1976).
ii Most agents listed in table IX, 1 have been tested in vitro where similar
t\ effects as in vivo have been demonstrated, with the exception of the

Table IX-1
The effect of physiological, pathological and pharmacological factors on the 5'-deiodina-
tion process in the peripheral conversion of T 4 (inhibition - , no effect 0, stimulation +;
* animal data only).

PHYSIOLOGICAL FACTORS
1. age : fetus —
neonate +
elderly subjects (-) , :i
2. sex* : females (relative to males) - '~\
males (relative to females) + •=
3. weight : weight loss - , rj
obesity +
4. diet : fasting -
carbohydrates +

PATHOLOGICAL FACTORS
1. thyroid disease: hyperthyroidism +
hypothyroidism —
2. non-thyroidal disease, traumas, surgical procedures

PHARMACOLOGICAL FACTORS
1. propylthiouracil
2. carbimazole
3. corticosteroids
4. amiodaxone (Cordarone)
5. iodide
6. iopanoic acid (Bilijodon, Telepaque)
7. amidotrizoic acid (Urografin) o a
8. ioglycamic acid (Bilivistan)
9. ipodate (Oragrafin) o I
10.
11.
12.
13.
14.
propranolol
lithium
growth hormone
estrogens*
androgens* • i
 182
corticosteroids (chapter VIII. 1). The mechanism of action of these agents
has, in most cases, not satisfactorily been elucidated. The enhancing effect
of growth hormone is probably related to the anabolic action of the
hormone wHh its attendant need for more energy (Sato et al 1977). With
respect to amiodarone and the radiographic contrast agents, it has been
suggested that some similarity in the structure of these agents with the
thyroxine molecule may be of significance (Burger et al 1976; Biirgi et al
1976; Wu et al 1978), as has been demonstrated for other iodinated
benzene derivatives (Escobar del Rey and Morreale de Escobar 1962) and
thyroid hormone analogs (Larson and Albright 1961; Pittman et al 1964).
Visser (1979) has recently clarified the intriguing phenomenon that, in
contrast to PTU, carbimazole has no effect on the conversion of T4. It is
proposed (cf. chapter I) that the intermediate sulphenyl iodide of the
enzyme (E-SI) is reduced by the cofactor to the native enzyme (E-SH), or
reacts with thioureylenes yielding mixed disulfides; it was found that the
methimazole-enzyme complex is more easily reduced by the cofactor.
The study of the pharmacological modulation of the peripheral
conversion of T4 has already provided a new agent for the treatment of
hyperthyroid patients: ipodate (Wu et al 1978).

IX.4. The peripheral conversion of TA and the pituitary-thyroid axis:

regulatory mechanisms.

The deiodination of T 4 is governed by two putative enzymes, 5'-

deiodinase and 5-deiodinase (chapter I). Physiological, pathophysiologi-
cal or pharmacological modulation of the peripheral conversion of T 4 very
often results in the 'low T3, high rT3 syndrome', which is caused by a
decreased T4-»T3 conversion rate and a decreased MCR of rT3, sometimes
associated with a small increase in the T4^rT3 conversion rate. Thus,
inhibition of the S'-deiodinating activity, rather than stimulation of the 5-
deiodinating activity is of great importance, and outer- and inner-ring
monodeiodination of T4 is not a random process as initially proposed by
Surks and Oppenheimer (1971). The factors that regulate the 5-
deiodination process are still obscure. The substrate and enzyme
concentration and the availability of non-protein sulfhydryl (SH) groups
in the tissues have been recognized as important regulators of the 5'-
deiodination — i.e. the conversion of T4-»T3 and rT3-»3,3'-T2. The decrease
in the S'-deiodinating activity has, in general, been explained by a lower
substrate concentration in hypothyroidism, by a lower concentration of
the enzyme in the fetus and in females, and by a lower concentration of SH-
groups during starvation and non-thyroidal illness (cf. chapter V.4). It is
assumed that the 5-deiodinase is le??. sensitive to lower concentrations of
reduced glutathione than the S'-deiodinase. Undoubtedly more regulatory
pathways for the deiodination of T4 will be recognized in the near future. It
 183
is intriguing that oral glucose, in contrast to intravenous glucose, is much
more effective in the restoration of the 'low T3, high rT3 syndrome'
(Westgren et al 1977; Burr et al 1979). Factors from the gut
(gastrointestinal hormones?) may therefore participate in the regulation of
the deiodination of T4.
t
The 'low T3, high rT3 syndrome' is not without consequences for the
setting of the pituitary-thyroid axis. This is best demonstrated by the rise in
plasma TSH secondary to pharmacologically induced S'-deiodination
inhibition in L-T4 treated subjects (cf. chapter VII). A slight transient
increase in TSH was also observed in the patients with acute myocardial
infarction (chapter VI), but in a more heterogeneous group of patients with
various non-thyroidal diseases no changes in the TSH levels or in the TSH
response to TRH could be detected (chapter V). It is therefore surprising
that a more pronounced increase in plasma TSH is not seen in patients
with a non-thyroidal illness, in whom the plasma T 3 concentration
decreases to a very low or even undetectable level. Apparently there is an
adaptation in the set point of the pituitary thyrotrophic cell for the release
of TSH. In theory, this could be done by modulation of the release of
TRH, or of the receptors on the thyrotroph, and also by a more efficient
intrapituitary monodeiodination of T 4 to T3 (cf. the studies of Larsen and
co-workers, described in chapter V.4). An altered sensitivity of the
pituitary thyrotroph has been reported in iodine deficiency (Chopra et al
1978), and also in the fasting state, where a blunted TSH response to TRH
is observed by most authors (Portnay et al 1974; Croxson et al 1977;
Carlson et al 1977; Azizi 1978; Burman et al 1979).

There are problems with thyroxine when the conversion of T4 is

changed. In some patients with non-thyroidal disease, plasma T4 is found
to be low, which is probably due to a decreased binding capacity of the
plasma proteins for thyroid hormones (cf. chapter V). However, in a
homogeneous group of patients with acute myocardial infarction we
observed a transient rise in plasma T4, presumably caused by some TSH-
induced thyroidal release of T4 and by impaired degradation of T4 (chapter
VI). A lowered MCR of T4 due to impaired deiodination is again strongly
suggested by the rise in T4 in the L-T4 treated subjects with
pharmacologically-induced inhibition of the S'-deiodination of T4 (chapter
VII). Although the data in the literature are rather confusing, higher
plasma T4 levels have been reported when the S'-deiodination is inhibited
(by fasting — Ingbar and Galton 1975, by PTU — Escobar del Rey and 1
Morreale de Escobar 1961; Slingerland and Burrows 1962; Hershman
1964; Frumess and Larsen 1975; for other agents see chapter VII.4) and
lower plasma T4 levels have been demonstrated when the 5'-deiodination is
activated (by growth hormone — Sato et al 1977, by polychlorinated
 184
biphenyls — Bastomsky et al 1976, or by estradiol benzoate — Chen and
Walfish 1978). Also, the clearance of radioactive PBI or T4 is reported to
be delayed by PTU (Oppenheimer et al 1972) and during fasting (Ingbar
and Galton 1975; Grant et al 1978), while an increased MCR of T4 has been
noted in obesity (Felt et al 1976).
In our opinion many cases of the so-called "T4-toxicosis" (a condition
not infrequently encountered in elderly subjects) are due to co-existent
non-thyroidal disease, the relatively low T3 and the high T4 plasma levels
being caused by inhibition of the 5'-deiodination of T4 (cf. chapter IV. 4c).

IX.5. The biological significance of the peripheral conversion of T4.

It is clear from the fore-going discussion that the extrathyroidal

deiodination of T4 into either T3 or rT3'is not a random process, but that
two independent although related mechanisms are involved. It is clear that
modulation of the peripheral conversion of T4 is a common feature of non-
thyroidal disease, and is qualitatively and quantitatively related to the
severity of the disease (chapters V and VI); it constitutes a basic response
of the body to stress. The existence of the 'low T3, high rT3 syndrome' in
'sick euthyroid patients' suggests that the shift in the conversion of T4
(resulting in less metabolically active T3 and more metabolically inactive
rT3) is an adaptive mechanism, by which the body seeks to protect itself
against excessive catabolism at a time when anabolism is challenged. The
question then arises whether, under these circumstances, some sort of
'hypothyroidism' exists at the level of the peripheral tissues, not
withstanding the fact that the functional state of the hypothalamic-
pituitary-thyroid axis is indicative of euthyroidism.
If the teleological points raised above are valid, rT3 should have less
catabolic activity than T3. Chopra et al (1978) have demonstrated in the
rat, that rT3 administration is without effect on body growth, urinary urea
nitrogen and plasma levels of T4, T3 and TSH. In contrast, T3 medication
resulted in an increase of urinary urea nitrogen and in a decrease of body
growth; thus, T3 exerts a catabblic effect as opposed to rT3. In another
series of experiments in rats, Cho.pra et al (1978) compared the effect of T4
treatment to that of treatment with T4 together with sodium ipodate
(ipodate strongly inhibits the T4-*T3 conversion). T4 alone increased
plasma T3 and urinary urea nitrogen and decreased body grov/th as
compared to controls, but the concomitant administration of ipodate
completely abolished the effects of T4, although plasma T4 had increased
dramatically because of the decrease in deiodinative degradation. These
results constitute strong evidence that in these experiments T4 in itself has
minimal catabolic action, and that the catabolic effects are mainly due to
the generation of T3 from T4. Further evidence that modulation of the
conversion of T4 has homeostatic significance comes from starvation

-r~r
 185
experiments. Gardner et al (1979) studied seven volunteers who fasted for
80 hours at two occasions: first without T3 replacement, and subsequently
with a 'physiological' replacement dose of T3. Serum T4 did not change and
serum rT3 increased in the two fasting periods, but the decrease in serum T3
was prevented in the T3 fast. The excretion of urea was 9.1% higher during
the T3 fast. They conclude that the decrease in serum T3 during fasting
spares muscle protein. A similar conclusion is reached by Carter et al
(1975).
Studies in patients with anorexia nervosa have also demonstrated
hypothyroid-like alterations in the peripheral tissues. The Achilles reflex
half-relaxation time in anorectic patients with a subnormal plasma T3 is
longer than in those with a normal T3 (Croxson and Ibbertson 1977). The
observed lowering of the androsterone/etiocholanolone ratio — due to
decreased 5 a reductase — (Bradlow et al 1976) and the elevated
tetrahydrocortisol/tetrahydrocortisone ratio — due to decreased 11-
hydroxysteroid dehydrogenase — (Boyar et al 1977) in anorexia nervosa,
are abnormalities in the androgen and cortisol metabolism also found in
hypothyroid patients (Hellman et al 1959; 1961). In patients with anorexia
nervosa the abnormal ratio's revert to normal when the patient is treated
with T3, which suggests that the abnormalities are due to the 'low T3
syndrome' caused by the weight loss per se. Interestingly, a reduced
androsterone/etiocholanolone ratio associated with the 'low T3 syndrome'
is also observed in lung cancer—where it is known to be a poor prognostic
sign — (Ratcliffe et al 1978), and an increase in the tetrahydrocortisol/
tetrahydrocortisone ratio is one of the nonspecific consequences of illness
(Zumoff et al 1973).
The situation with respect to the presumably enhanced 5'-deiodination
of T4 in obesity is less clear. It has been reported that the body can adapt to
caloric restriction by decreasing its energy needs and by becoming
metabolically more efficient; these changes partially offset the expected
weight loss in obese subjects (Bray 1969; Grant et al 1978). On the other
hand, it is difficult for normal subjects to gain much weight by overeating.
The debate on increased thermogenesis in overnutrition is continuing since
1900. The generation of extra heat in the face of increased food intake (the
old concept of'Luxuskonsumption') may play a role in the maintenance of
a stable body weight, and is possibly induced by an increase in plasma T3
levels (Sims 1976; Davidson and Chopra 1979). Subjects with morbid
obesity may be limited in their ability to produce heat; their reduced
thermogenesis seems to be constitutive rather than the result either of
developmental or other environmental factors (Bray et al 1976; James and
Trayhurn 1976; Bray 1977; Jung et al 1979).
The significance of the low T3 and high rT3 plasma concentrations in
fetal life is unknown, but could be related to the energy metabolism of the
fetus. In this respect it is of interest that in monkey hepatocarcinoma cells
 186
the T4-»rT3 pathway is very much enhanced and the T4-iT3 pathway
strongly inhibited (Sorimachi and Robbins 1977, 1978). It has been
speculated that this represents dedifferentiation to a more immature cell
line resembling fetal hepatocytes. The striking resemblance between the
fate of rT,and that of a M-fetoprotein (an acute phase protein that inhibits
the inflammatory reaction, found in high concentrations in the plasma of
the fetus, in cord blood, after injury and during liver carcinogenesis)
further underlines the basic significance of the conversion of T4.
It thus appears that modulation of the conversion of T4 is an important
homeostatic mechanism of the body in the regulation of its energy needs.
But, as Tweedledee said: "If it was so it might be; and if it were so, it would
be; but as it isn't, it ain't. That's logic".
It has been suggested that the monodeiodination of T4 to T3 is essential
for the action of T4, and that T4 itself possesses little or no biological
activity, T3 being the active thyroid hormone (among others, Oppenheimer
et al 1972; Frumess and Larsen 1975). Thus viewed, the function of T4 is
merely a prohormone of T3. Chopra et al (1973) have argued against this
proposition because
1) a normal plasma T3 concentration in the presence of a low plasma T4 is
not sufficient to sustain euthyroidism,
2) a supranormal plasma T3 concentration is required in T3-treated
hypothyroid patients to maintain euthyroidism, and
3) despite a low plasma T3 concentration, the fetus is assumed to be
euthyroid. However, in the light of the recent knowledge on the
intracellular T4-»T3 deiodination and the binding of T3 to nuclear receptors
the validity of these arguments is now questionable. In this respect, the
studies of Larsen and co-workers (cf. chapter V.4) are especially
important, as they have shown that the intrapituitary T4-»T3
monodeiodination generates half of the nuclear T3 in the thyrotropic cell,
the other half being derived from plasma T3. Studies from the Escobar's
have also repeatedly demonstrated the importance of the T4-»T3 conversion
in vivo in the expression of T4 activity (Hervas et al 1976; Obregon et al
1979). Although it cannot be denied that T4 may have some intrinsic
activity (du Breuil and Galton 1978; Chopra et al 1978; Surks and
Oppenheimer 1978) it seems to us that thyroid hormone activity in the
tissues is expressed mainly by T3. This is not to say that T4 has no functions
other than that of a prohormone. The significance of its binding tc nuclear
receptors and its occupancy of receptor sites has so far not been elucidated.

IX.6. Epilogue.

The rapid development of endocrinology has deepened our insight in

hormonal mechanisms, but also has brought confusion because classical
concepts have been undermined. For instance, the very concept of a
 187
hormone is now unclear since agents that transmit signals by endocrine,
paracrine or neurocrine mechanisms are all referred to as hormones. And
what is synthesized within an endocrine cell, is not necessarily the same
product that leaves the cell (e.g. preproparathormone, proparathormone
and parathormone), and what is secreted by an endocrine cell into the V:
bloodstream is not necessarily the same product that ultimately provokes
the hormonal effect in the peripheral tissues (e.g. testosterone and 6.
dihydrotestosterone). And the hormonal effects in the target cells may be
modulated by the receptor state (e.g. 'up' and 'down' regulation of insulin
receptors), not to mention the possibiliy of post-receptor alterations
(Hoffenberg 1979).
The fate of thyroxine is an excellent illustration of these points. Firstly,
thyroxine synthesized in the thyroid gland may be converted
intrathyroidally to T3 as has been demonstrated in animals (Bidey et al
1976; Mindroiu et al 1976; Green 1978; Laurberg 1978, 1979). Secondly,
thyroxine is deiodinated in the peripheral tissues into either the potent
hormone T3 or the metabolically inactive rT3. And thirdly, the receptors
for thyroid hormones can be altered - fasting induces a decrease in nuclear I-"
T3 receptors (DeGroot et al 1977; Schussler and Orlando 1978) —, but
thyroid hormones can also modulate the receptors of other hormones:
thyroid hormone excess induces an increase of glucagon receptors
(Madsen and Sonne 1976), and the number of /J-adrenergic receptors of
skeletal muscle, heart, fat cells and erythrocytes is increased in
hyperthyroidism and decreased in hypothyroidism (Kunos et al 1974;
Banerjee and Kung 1977; Ciaraldi et al 1977; Kunos 1977; Williams et al
1977; Giudicelli 1978; Malbon et al 1978; Sharma and Banerjee 1978;
Bilczikian et al 1979; Popovic et al 1979). It thus appears that the
peripheral tissues have the capability to modulate the expression of the
hormonal effect to a great extent, by the regulation of the amount of T3
generated from T4 and by the regulation of the affinity and capacity of the
thyroid hormone receptors.
Further study of these mechanisms in the target cells is therefore needed
to establish the real significance of the hypothyroid-like alterations
observed in the 'low T3, high rT3 syndrome' (Wiersinga and Touber 1978).
It may even be necessary to redefine the concepts of hyperthyroidism and
of hypothyroidism.
%

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I
k1

SUMMARY

The peripheral conversion of the thyroid hormone tetraiodothyronine

or thyroxine (T4) into triiodothyronine (T3) and reverse triiodothyronine
(rTj) is defined as the extrathyroidal deiodination of T4, by which -
"I depending on the site of removal of an iodine atom- either the
S i
metabolically very potent hormone T3 or the metabolically inactive rT3
arises {chapter I). This deiodination process generates the bulk of the daily
production of T3 and rT3, and is a major pathway in the degradation of T4.
The conversion of T4 has been demonstrated in a wide variety of tissues;
the greatest deiodination capacity is found in the liver and kidneys. The
deiodination of T4 is enzymic in nature, and sulfhydryl groups serve as
cofactor; the putative enzyme 5'-deiodinase promotes the T4 -• T3
conversion, and the 5-deiodinase the T4 -• rT3 conversion. The main goal of
this study was to delineate several physiological, pathological and
pharmacological factors involved in the modulation of the peripheral
conversion of T4.

The assays used in this study are described in chapter II. Thyrotropin
(TSH) and the iodothyronines rT3, T3 and T4 were determined in plasma
by radioimmunoassay (the method of Murphy and Pattee was initially
applied for the assay of T4). The triiodothyronine uptake ratio (T U) was
measured by the Triosorb-M 125 kit. The normal values of these t. sts, as
determined in 63 euthyroid volunteers, are presented in chapter HI.
Median values (in brackets the inner limits for the percentiles P2.5 and
P97.5) are: TSH 1.1 ( < 0.3-3.l)mU/l, rT3 0.22(0.14-0.38)nmol/l,T31.75
(1.30-2.45) nmol/l,T4101 (75-135) nmol/l,T3U 0.93 (0.81-1.07), FT4 index
95 (70-122). No effect of sex or age was found, although a tendency to
lower T3 and higher rT3 values was observed in old age.
The TSH response to 200//g TRH i.v. was lower in males and in subjects of
60 years and older as compared to females and younger subjects. The
median peak TSH value was 8.5 mU/1 (inner limits 2.8-22.0 mU/1). The
normal T3 response to TRH was defined as an absolute increment of ^>0.55
nmol/1 and a relative increment of > 30%.
In 19 cord blood samples from healthy newborns plasma T3 was low and
plasma rT3 very high.

Abnormal thyroid function tests were found in 125 patients suspected of

thyroid disease {chapter IV). According to the test results, they were
 195
classified into 7 groups: overt hyperthyroidism (supranormal FT4 index
and T3, subnormal TSH response; n=55), mild hyperthyroidism or T3-
toxicosis (normal FT4 index, supranormal T3, subnormal TSH response;
n=l 1), subclinical hyperthyroidism (normal FT4 index and T3, subnormal
TSH response; n = 15), subclinical hypothyroidism (normal FT 4 index and rf;
T3, supranormal TSH response; n=6), mild hypothyroidism (subnormal
FT4 index, normal T3, supranormal TSH response; n=6), overt hypo-
thyroidism (subnormal FT, index and T3, supranormal TSH response;
n=27), and T4-toxicosis (supranormal FT4 index, normal T3, subnormal
TSH response; n=S). It is concluded that hyperthyroidism and
hypothyroidism are graded phenomena, and that the TSH response to
YRH is the most sensitive test in the detection of abnormal thyroid
function. The finding of a normal TSH response virtually excludes
hyperthyroidism, but a sub-normal TSH response is also found in other
(especially psychiatric) disorders, sometimes associated with a normal T3
response. The biochemical pattern of T4-toxicosis is in most cases caused
by impaired S'-deiodination of T4.
From the observed ratio's rT3/T3, rT 3 /T 4 and T 3 /T 4 it is speculated that
there is an overproduction of rT3 relative to T3 in hyperthyroidism, and of
T3 relative to rT3 in hypothyroidism.
li
In 20 patients with various acute non-thyroidal diseases, a subnormal T3
. was found in 84%, a supranormal rT3 in 63%, and a subnormal T4 in 32%
(chapter V). The plasma T3 and rT3 values slowly returned into the normal
range if clinical recovery was delayed, whereas a further decrease of T3 and
increase of rT3 was observed in the patients who died; this suggests that the
severity of a disease is qualitatively related to the changes in the conversion
of T4. The subnormal T4 values are caused by a decreased binding capacity
of the plasma proteins for thyroid hormones, possibly with involvement of
an inhibitor for thyroid hormone binding. Despite the substantial decrease
• in plasma T3 concentrations the TS H levels were not elevated, nor were the
; TS H- and T3-responses to TRH different on the day of admission and two
f; weeks later. The discrepancy between some sort of 'hypothyroidism' in the
£'. peripheral tissues and the 'euthyroidism' at the central pituitary-thyroid
[1 level may be explained by the capability of the pituitary thyrotroph to
'4 derive its nuclear T3 from local T4 conversion to a much greater extent than
< the peripheral tissues.

i;< A quantitative relationship between the severity of a disease and the

changes in the conversion of T4 was found in 34 patients with acute
f. myocardial infarction (AMI) (chapter VI). The fall in T3 and the rise in rT3
| were greater in patients with a complicated AMI than in those with an
| uncomplicated AMI, and a linear correlation was found between the
V infarct size (as estimated by the peak SGOT value) and the increment of

- - - - - - ••-••.-.>--;,r
 196
rT3, the decrement of T3, and the highest observed rT 3 /T 3 and rT 3 /T 4
ratio's. Plasma T4 levels peaked approximately one week after the onset of
AMI, probably due to a decreased degradation of T4 and to a slight TSH-
mediated increase in thyroidal secretion provoked by the inhibited 5'-
deiodination of T4. p..

The effect of the B-adrenoceptor blocking agent propranolol on the

conversion of T4 was studied in 11 hyperthyroid patients and in 6
hypothyroid patients on L-T4 substitution therapy (chapter VII). Plasma
T3 decreased and plasma rT3 increased in both groups under propranolol
medication, whereas T4 and TSH increased in the hypothyroid patients.
These changes can be explained by an inhibitory effect of propranolol on
the 5'-deiodination of T4. The effect of propranolol on the in vitro
conversion of T4 into T3 using isolated rat liver parenchymal cella is
described in chapter VIII. Propranolol lowers the conversion ratio when
added in concentrations of 580 and 1160//M, but has no inhibitory effect
when 290//M is added.

It thus appears that physiological, pathophysiological, or pharmaco-

logical modulation of the peripheral conversion of T4 very often results in
the 'low T3, high rT3 syndrome' (chapter IX). As demonstrated in kinetic
studies by others, the syndrome is caused mainly by a decreased PR of T 3
from T4 and by a decreased MCR of rT3, both due to a decrease in the 5'-
deiodinating activity. This decrease has, in general, been explained by a
lower substrate concentration in hypothyroidism, by a lower concentra-
tion of the enzyme in the fetus and in female rats, and by a lower
concentration of sulfhydryl groups during starvation and in non-thyroidal
illness. Inhibition of the S'-deiodination of T4 not infrequently results in a
decreased MCR of T4, and in an increased release of TSH. In general,
however, the pituitary-thyroid axis functions as if an euthyroid state exists,
despite hypothyroid-like alterations in the peripheral tissues. The
biological significance of the iow T3, high rT3 syndrome' is presumably
related to the energy metabolism of the organism: the shift in the
conversion of T4 can be interpreted as an adaptive mechanism, by which
the body seeks to protect itself against excessive catabolism at a time when
anabolism is challenged. The peripheral tissues thus have the capability to
modulate the expression of the hormonal effect to a great extent by the
regulation of the amount of T3 locally generated from T4.
k
I
SAMENVATTING %
D'
De perifere conversie van het schildklierhormoon tetrajodothyronine of j
thyroxine (T4) in trijodothyronine (T3) en 'reverse' trijodothyronine (rT3)
wordt gedefinieerd als de dejodering van T4 buiten de schildklier, waarbij
-afhankelijk van de plaats waar een jodiumatoom wordt verwijderd - het
metabool sterk werkzame hormoon T 3 of het metabool inactieve rT3
ontstaat (hoofdstuk I). Deze dejodering van T4 levert verreweg het grootste
aandeel in de dagelijkse productie van T3 and rT3, en is een kwantitatief :
belangrijke weg in de afbraak van T 4 . De omzetting van T4 is aangetoond i
in uiteenlopende weefsels; de lever en de nieren hebben het grootste ; I
dejoderend vermogen. De dejodering van T4 is een enzymatisch proces, •
waarbij SH-groepen fungeren als co-factor; het hypothetische enzym 5'- i
\ dejodinase bevordert de omzetting van T4 in T3, het S-dej odinase van T4 in í
: rT3. Het belangrijkste doel van deze studie was om factoren van
: fysiologische, pathologische en farmacologische aard die van invloed zijn
• op de conversie van T4, nader te leren kennen.
De gebruikte bepalingsmethodieken worden beschreven in hoofdstuk
IL Het schildklier-stimulerend-hormoon TSH en de jodothyronines rT3,
T 3 en T4 zijn langs radioimmunologische weg bepaald in plasma (voor
de bepaling van T4 is aanvankelijk de methode van Murphy-Pattee
toegepast). De T3-(hars)opname (T3U) is bepaald met behulp van de :
Triosorb-M 125 kit. De normaalwaarden voor deze testen, vastgesteld bij
63 euthyreote vrijwilligers, zijn vermeld in hoofdstuk III. De médiane
:. waarden, met tussen haakjes de (waarschuwende) grenzen voor de
! percentielen P 2 . 5 en P97.5, zijn: TSH 1.1 (< 0.3-3.1) mU/1, rT3 0.22 (0.14- i
| 0.38) nmol/l,T 3 1.75 (1.30-2.45) nmol/l,T 4 101 (75-135) nmol/l,T 3 U 0.93 \ \
\- (0.81-1.07), FT4 index 95 (70-122). Leeftijd en geslacht waren niet van '1 j
[i invloed op de uitslagen, hoewel er een tendens bestond tot lagere T, en : ;
\\ hogere rT3 waarden bij mensen van 60 jaar en ouder. j )
{! De TSH response op 200 //g TRH i.v. was bij mannen lager dan bij
| vrouwen, en bij personen van 60 jaar en ouder lager dan bij jongeren. De
'f médiane waarde van de TSH-piek was 8.5 mU /1 (waarschuwende grenzen fi
i 2.8-22.0 mU /1). De normale T3 response op TRH werd gedefinieerd als een |
| absolute toename van > 0.55 nmoï /1 en een relatieve toename van > 30%. j
i In het navelstrengbloed van 19 gezonde pasgeborenen was het plasma T3 -\
I laag en het plasma rT3 erg hoog. j |;
l < f
 •tr-j-

198
Afwijkende uitslagen van de schildklierfunctietesten werden gevonden
bij 125 patiënten die verdacht werden van een gestoorde schildklierfunctie
{hoofdstuk IV). Op grond van de testresultaten waren 7 groepen te
onderscheiden: manifeste hyperthyreoïdie (supranormale FT4 index en T3,
subnormale TSH response; n=55), lichte hyperthyreoïdie of T3-toxicosis
(normale FT4 index, supranormaal T3, subnormale TSH response; n=l 1),
subklinische hyperthyreoïdie (normale FT4 index en T3, subnormale TSH
response; n=15), subklinische hypothyreoïdie (normale FT4 index en T3.
supranormale TSH response; n=6), lichte hypothyreoïdie (subnormale
FT4 index, normaal T3, supranormale TSH response; n=6), manifeste
hypothyreoïdie (subnormale FT4 index en T3, supranormale TSH
response; n=27), en T4-toxicosis (supranormale FT4 index, normaal T3,
subnormale TSH response; n=5). Geconcludeerd wordt, dat hyper-
thyreoïdie evenals hypothyreoïdie een gegradueerd verschijnsel is, en dat
de TSH response op TRH de gevoeligste indicator is voor het opsporen
van een afwijkende schildklierfunctie. Een normale TSH response op
TRH sluit het bestaan van een hyperthyreoïdie vrijwel uit, maar een
subnormale tot afwezige TSH response komt ook voor bij andere (vooral
psychiatrische) ziektebeelden, waarbij de T3 response dan nog normaal
kan zijn. Het biochemische patroon van een T4-toxicosis is in de meeste
gevallen te verklaren uit een remming van de 5'-dejodering van T4.
Op grond van de waargenomen ratio's rT3/T3, rT 3 /T 4 en T3/T4 lijkt het
niet onmogelijk, dat bij hyperthyreoïdie relatief meer rT3 dan T3 wordt
geproduceerd, en bij hypothyreoïdie relatief meer T3 dan rT3.

Bij 20 patiënten met verschillende acute niet aan de schildklier

gerelateerde ziektebeelden werd een subnormaal T3 gevonden in 84%, een
supranormaal rT3 in 63%, en een subnormaal T4 in 32% (hoofdstuk V). De
plasma T3 en rT3 waarden keerden vrij langzaam tot in het normale gebied
terug indien het klinisch herstel traag verliep, terwijl een verdere afname
van het T3 en een toename van het rT3 werd gezien bij patiënten die
overleden; dit suggereert een kwalitatief verband tussen de ernst van een
ziekte en de veranderingen in de conversie van T4. De subnormale T4
waarden zijn te verklaren uit een afgenomen bindingscapaciteit van de
plasma-eiwitten voor schildklierhormoon, waarbij mogelijk ook een
remmer van deze binding betrokken is. Ondanks de aanzienlijke verlaging
van het plasma T3 gehalte zijn de TSH spiegels niet verhoogd; de TSH- en '•%
Tj-responsies op TRH op de dag van opname verschillen niet van de
responsies twee weken later. De discrepantie tussen 'hypothyreoïdie' in de 'ii
perifere weefsels en 'euthyreoïdie' in de hypofyse-schildklier as is wellicht
te verklaren uit het vermogen van de hypofysaire TSH-producerende cel
om zijn aan de kern gebonden T3 in een veel sterkere mate te ontlenen aan
de plaatselijke omzetting van T4 dan de perifere weefsels.
 19S
Een kwantitatief verband tussen de ernst van een ziekte en de

s veranderingen in de conversie van T4 kon worden aangetoond bij 34

patiënten met een acuut hartinfarct (hoofdstuk VI). De daling van T3 en de
stijging van rT3 waren bij patiënten met een gecompliceerd verlopend
hartinfarct groter dan bij patiënten met een ongecompliceerd verloop. Er
bestond een lineair verband tussen de infarctgrootte (v ¿chat aan de hand
van de hoogste SGOT waarde) en de toename van rT3, de afname van T3,
en de hoogst waargenomen rT3/T3 en rT3/T4 ratio's. De plasma T4
waarden stegen tot een maximum ongeveer 7 dagen na ontstaan van het
infarct; deze stijging is waarschijnlijk het gevolg van een verminderde
afbraak van T4 en een geringe door TSH gestimuleerde toename in de
schildkliersecretie, beide veroorzaakt door remming van de S'-dejodering
van T4.

Het effect van de ß-blokker propranolol op de conversie van T4 werd

bestudeerd bij 11 thyreotoxische patiënten en bij 6 hypothyreotische
patiënten op L-T4 substitutie (hoofdstuk VII). Het plasma T 3 daalde en het
plasma rT3 steeg in beide groepen tijdens toediening van propranolol,
terwijl het T4 en het TSH stegen in de patiënten met hypothyreoïdie. De
veranderingen zijn te verklaren uit een remmend effect van propranolol op
de S'-dejodering van T4. Het effect van propranolol op de in vitro
omzetting van T4 in T3 (met gebruikmaking van geïsoleerde I
leverparenchymcellen van ratten) wordt beschreven in hoofdstuk VIII.
Propranolol toegevoegd in een concentratie van 580 en 1160^/M verlaagt
de conversie, maar toevoeging van 290 f*M heeft geen invloed op de
conversie.

Fysiologische, pathophysiologische of farmacologische beïnvloeding

van de perifere conversie van T4 resulteert dus dikwijls in het
'laag T3, hoog rT3 syndroom' (hoofdstuk IX). Zoals uit kine-
tisch onderzoek door anderen verricht blijkt, wordt dit syndroom
hoofdzakelijk veroorzaakt door een verminderde productie van T3 uit T4
en door een verminderde metabole klaring van rT3, beide als gevolg van
een afname in de S'-dejodering. Deze afname is - in grote lijnen - te
verklaren uit een lagere substraatconcentratie bij hypothyreoïdie, uit een
lagere concentratie van het enzym bij de foetus en bij vrouwtjesratten, en
uit een lagere concentratie aan sulfhydryl-groepen tijdens vasten en bij
ziekten in het algemeen. Remming van de S'-dejodering van T4 leidt niet
zelden tot een verminderde metabole klaring van T4 en een toegenomen
TSH secretie. Doorgaans functioneert de hypofyse-schildklier as echter
alsof er sprake is van euthyreoïdie, ondanks de hypothyreoïdie-achtige
veranderingen in de perifere weefsels. De biologische betekenis van het
'laag T3, hoog rT3 syndroom' houdt vermoedelijk verband met de
energiehuishouding van het lichaam: de veranderingen in de conversie van

ï
é
kr.
200
T4 zijn te interpreteren als een aanpassingsmechanisme, waarmee het
lichaam zichzelf kan beschermen tegen overmatig katabolisme ten tijde
van een toch al moeizaam anabolisme. De perifere weefsels lijken derhalve
over het vermogen te beschikken het hormonale effect in aanzienlijke mate

I te beinvloeden door regulatie van de hoeveelheid ter plaatse uit T4

gevormd T3.

i-.
I,-
f•

4
 SAMENVATTING VOOR DE LEEK ;

De Nobelprijs voor geneeskunde is in 1977 toegekend aan Rosalyn ¡

;
Yalow voor haar baanbrekend werk bij de ontwikkeling van de
radioimmunologische bepaling van hormonen, en aan Roger Guillemin en
Andrew Schally voor de isolering van stoffen uit de hersenen die de afgifte
van hormonen door het hersenaanhangsel (de hypofyse) reguleren (met
name voor de isolering van het TRH of het TSH-releasing-hormone, een
hormoon dat de afgifte van TSH door de hypofyse stimuleert; TSH is het, \
schildklierstimulerend hormoon uit de hypofyse). Zonder hun pioniers- ; ,
werk zouden de studies in dit proefschrift niet mogelijk zijn geweest.
De schildklier produceert twee hormonen, T4 en T3, waarvan het T3 ;
eigenlijk niet goed meetbaar was door de bijzonder lage concentratie waar- \
in het in het menselijk bloed voorkomt. Met behulp van de door Yalow en
Berson ontwikkelde radioimmunologische methodiek is de bepaling van '•'• |
T 3 sinds het begin het begin van de zeventiger jaren veel eenvoudiger en . |
betrouwbaarder geworden.
Schildklierziekten uiten zich vaak doordat teveel of te weinig
schildklierhormoon wordt geproduceerd; men spreekt dan van
hyperthyreoïdie of hypothyreoïdie, waarbij de T4 en T3 concentraties in het
bloed verhoogd of verlaagd zijn. Na de invoering van de T3 bepaling bleken
er patiënten te zijn bij wie het ziektebeeld van de hyperthyreoïdie
:
uitsluitend veroorzaakt werd door een overmatige T3 productie (de zgn.
T3-toxicosis). Dit beeld kon in het verleden, toen de T3 bepaling nog niet
: beschikbaar was, en doordat het T4 gehalte in het bloed hierbij normaal is,
I vaak moeilijk herkend worden. Uit ons onderzoek blij kt dat de Tj-toxicosis •
S niet zeldzaam is: het kwam voor in 17% van de patiënten met ! >
SÏ hyperthyreoïdie. ] jj_
g Ook de invoering van TRH-test in het begin van de zeventiger jaren |'
[| heeft de diagnostiek van schildklierziektes sterk verbeterd. Men geeft |:
;| hierbij een injectie van TRH in de ader, en meet vervolgens de reactie van • '• !!
| TSH in het bloed; normaal stijgt het TSH gehalte na toediening van TRH. '| , b
| De TSH afgifte wordt echter ook beïnvloed door T3 en T4: een te lage '} 1
1 concentratie schildklierhormoon stimuleert de TSH afgifte, een te hoge i t\
\ concentratie schildklierhormoon remt de TSH afgifte. Bij hyperthyreoïdie | ^
blijkt de TSH response op TRH verminderd tot afwezig te zijn, bij f i
hypothyreoïdie verhoogd. Het grote belang van de TRH test ligt in het feit, ,'j ' |>
dat een normale TSH response het bestaan van een hyperthyreoïdie vrijwel i ¿;
uitsluit. Zoals ook uit ons onderzoek blijkt, is de gevoeligheid van deze test \ ffe
 -BV

• 202 V;
i : it
v in de diagnostiek van een gestoorde schildklierfunctie zelfs zo groot, dat '*£•
£ niet zelden reeds een afwijkende TSH response wordt gevonden wanneer 'j£.
f de T 3 en T 4 gehaltes in het bloed (nog) normaal zijn en de patiënten geen |%
£ klinische verschijnselen van een teveel of een tekort aan schildklierhor- fe
| moon tonen. Of een dergelijke'subklinische'hyper-of hypothyreoïdie ook ; ? |¿ :
l behandeld moet worden, is overigens de vraag. Wel lijken deze patiënten ' 'ix
een grotere kans te hebben op de ontwikkeling van een manifeste hyper- of ii
hypothyreoïdie. , |Ï;
;
Na het beschikbaar komen van de T3 bepaling vond men lage tot zeer p
lage T3 gehaltes in het bloed van patiënten met uiteenlopende |)
ziektebeelden die op zich niets met de schildklier te maken hebben. Het E
opvallende was, dat deze patiënten niet de indruk maakten een tekort aan f.
schildklierhormoon te hebben. Na genezing van de ziekte bleek het T3 t
gehalte in het bloed weer normaal te zijn. De verklaring voor dit í
intrigerende verschijnsel is als volgt. Het is de laatste jaren duidelijk i:
geworden dat het grootste deel van het dagelijks geproduceerde T3 niet - ; !: •
zoals men zou denken - gevormd wordt in de schildklier, maar afkomstig is : 'il
?
van de omzetting van T 4 in T 3 buiten de schildklier. Bij deze 'perifere
conversie' wordt van T 4 , dat vier jodiumatomen heeft, één jodiumatoom
afgesplitst, waardoor T 3 ontstaat. Uit studies van Chopra is voorts
gebleken, dat door afsplitsing van een jodiumatoom o p een andere plaats
in het T 4 molecuul een verwante verbinding met eveneens drie
jodiumatomen ontstaat (het spiegelbeeldige of 'reverse' T 3 , afgekort als
rT 3 ), dat echter in tegenstelling tot T 3 geen hormonaal effect heeft in de zin
van stimulatie der stofwisseling. Bij ziek zijn in het algemeen daalt nu het
T 3 gehalte en stijgt het rT 3 gehalte in het bloed door veranderingen in de
perifere conversie van T 4 . Herkenning van dit patroon is belangrijk, daar
het waarschijnlijk schadelijk is patiënten met een aldus ontstaan laag T 3
met schildklierhormoon te behandelen. Een tweede opvallend fenomeen
bij dit 'laag T 3 , hoog rT 3 ' beeld is, dat het TSH gehalte in het bloed hierbij
normaal is, en niet stijgt zoals bij patiënten met een laag T 3 t.g.v.
:
hypothyreoïdie wel het geval is. Wij vonden dat de TRH-test onder deze
omstandigheden bij acuut zieke patiënten zijn diagnostische waarde blijft
behouden. Verder namen wij waar, dat bij patiënten die ernstig ziek zijn en
|? zich slechts langzaam herstellen van hun ziekte, de T 3 gehaltes lager zijn en ¡
[-) dit ook langer blijven dan bij patiënten die minder erg ziek zijn. Bij
i
Hi patiënten die overleden, daalde het T3 voor de dood en steeg het rT3 nog
f| verder. Een kwantitatief verband tussen de ernst van een ziekte en de
H veranderingen in de conversie van T4 konden wij duidelijk aantonen bij Î
f patiënten met een acuut hartinfarct. De ernst van een hartinfarct is ¿f
i gerelateerd aan de grootte van het infarct. Wij vonden een rechtlijnig k
f verband tussen de grootte van het infarct en de mate van T3 daling en rT3 f
J§/-' stijging. '|
¥ Patienten met hyperthyreoïdie worden vaak behandeld met proprano- ;

• & .
 V 203 ' {•
••iv. i?
'•% lol, een middel dat de effecten van heel andere hormonen dan het . .t
>:
> schildklierhormoon blokkeert. Het was dan ook niet geheel duidelijk, %
fk waarom dit medicijn bij overmatige schildklierhormoonproductie vaak zo 'f
f-;;: goed helpt. Wij hebben kunnen aantonen dat propranoiol de conversie van .. f
\t T 4 beinvloedt, zodanig dat minder van het sterk werkzame hormoon T3 en ; ,. f
I' meer van het inactieve rT3 ontstaat. Dit verklaart althans ten dele het $
I therapeutisch effect ervan bij verhoogde schildklierwerking. De remmende : |i
I> werking van propranoiol op de vorming van T3 uit T4 hebben wij ook |'i
\.J aangetoond in een kunstmatig systeem van rattelevercellen. Voegt men •->
,
I. aan levercellen T4 toe, dan maken de levercellen T3 uit het toegevoegde T4;
;' dit is een fraai model voor bestudering van de T4 conversie in een
• reageerbuisje. Bestudering van de conversie van T4 heeft inmiddels al een
nieuw geneesmiddel voor de behandeling van hyperthyreoidie opgeleverd: '
\ een van de door Chopra en medewerkers onderzochte stoffen remt de \
;' conversie van T4 in T 3 zo sterk, dat de verschijnselen van teveel {
X schildklierhormoon erdoor verminderen. • I

Veranderingen in de perifere conversie van T4 treden op onder tal van

omstandigheden. Men heeft het iaag T3, hoog rT3' beeld gevonden bij het
ongeboren kind, bij een laag lichaamsgewicht, tijdens vasten, bij ziek zijn
in het algemeen, na operaties, en bij gebruik van diverse medicamenten. De
betekenis van deze veranderingen in de perifere conversie van T4 is
onduidelijk. Waarschijnlijk is het een aanpassingsmechanisme, recht-
streeks betrokken bij de energiehuishouding van het lichaam. Men kan
zich tenminste goed indenken dat bij ziekte, als de omstandigheden voor de
opbouwende processen in het lichaam ongunstig zijn, een overmatige
afbraak voorkomen kan worden door verlaging van de T 3 productie,
waardoor de stofwisseling en het energieverbruik van de weefsels wordt
verminderd. Recente experimenten bij vrijwilligers die vastten,versterken ;
dit vermoeden. Vasten leidt tot een dating in het T3-gehalte en tot eiwitaf-
braak (~ spierverlies). Voorkomt men de T3-daling door toediening \
van T3, dan is de eiwitafbraak groter dan zonder T3 toediening. Dit
betekent dus, dat de T3-daling bij vasten een eiwitsparend effect heeft. :
Het vermogen van veel organen om zelf T3 uit T4 te maken, en dit
mogelijk ook zelf te regelen, houdt in dat het hormonaal effect van
schildklierhormoon op deze organen goeddeels ter plaatse wordt bepaald, i
tot op zekere hoogte onafhankelijk van de schildklier. Dit inzicht heeft het \
klassieke endocrinologische denken in 'centrale' regelmechanismen
ingrijpend gewijzigd. •
 e

5>'
ir'- ABBREVIATIONS
tí'
- symbol for radioactivity; the labeled hormone
i Ab - antibody
- antigen
B.I
Ag
AMI - acute myocardial infarction
a-MPT -oi-methyl-p-tyrosine
AS - antiserum
b - bovine
B« - initial binding (the percentage of tracer bound to the antibody in the
first zero-standard point of the standard curve)
- percentage of tracer specifically bound to antibody
BSA - bovine serum albumin
CG - choriogonadotropin; chorionic gonadotropin
CHO - carbohydrates
c.p.m. - counts per minute
DA - double antibody
DASP - double-antibody solid phase
DPH - diphenylhydantoin
DTT - dithiothreitol
FD - final dilution
FFA - free fatty acids
FSH - follitropin; follicle-stimulating hormone
FT, - free triiodothyronine
FT4 - free thyroxine
FT 4 índex - free thyroxine index (T3U xT 4 )
GH - growth hormone
h - human
Ht - hematocrit
LH - lutropin; luteinizing hormone
MCR - metabolic clearance rate
MRC • Medical Research Council
PBS - phosphate buffered saline
PEG - polyethylene glycol
PR - production rate
PTÜ - propylthiouracil
RIA - radioimmunoassay
rT, - reverse triiodothyronine; 3,3',5'-triiodo-L-thyronine
SGOT - serum glutamic oxalacetic transaminase
SH - non-protein sulfhydryl groups
T3 - triiodothyronine; 3,5,3'-triiodo-L-thyronine
T3U - triiodothyronine uptake ratio
 205
- thyroxine; 3,5,3',5'-tetraiodo-L-thyronine
T 4 (D) • T4-assay by displacement, the competitive protein binding method of
Murphy-Pattee
TBG - thyroxine binding globulin •_ - ' . _ . _z _._-._•
TBP - thyroid hormone binding proteins
TBPA - thyroxine binding pre-albumin -- . ~.
TETRAC - tetraiodothyroacetic acid
TRH - thyroliberin; thyrotropin-releasing hormone; protirelin
TRIAC - triiodothyroacetic acid
TSH - thyrotropin; thyrotropic hormone