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Simple screening test for the evaluation of thyroid function
113
CLINICA CHIMICA ACTA
SIMPLE
SCREENING
TEST
FOR THE
EVALUATION
OF THYROID
FUNCTION (EXPERIENCE BASED
WITH
ON RESIN
TWO IN-VITRO
UPTAKE
;1311]THYROID
AND SEPHADEX
FUNCTION
TESTS
GEL FILTRATION)
SUMMARY
A modification of the [1311]tri-iodothyronine (TJ resin uptake test was investigated. A simple, fast and convenient method was elaborated, eliminating several time-consuming steps in the procedure, such as pre-incubation of the sample, preweighing of resin, cumbersome washing procedure, furthermore choosing convenient temperature conditions (handling at room temperature, incubation at 37’) and limiting the incubation time and the number of radioactivity measurements needed. The test can be carried out in a routine isotope laboratory, without special equipment. The results of the test were compared with a Sephadex gel filtration T8 test and, in the case of suspected thyroid disorders, also with the protein-bound iodine (PBI) and the [1311]thyroid uptake test in vitro. With each test (resin uptake and gel filtration), most patients can be classified correctly as euthyroid, hyperthyroid and hypothyroid ones, according to the clinical diagnosis. However, values in the hypothyroid range are found during pregnancy and under the influence of oral contraceptives, whereas gross abnormalities in serum protein pattern can give rise to values in the hyperthyroid range.
INTRODUCTION
After secretion into the blood, the thyroid hormones thyroxine (TJ and tri-iodothyronine (T,) are, for the most part, bound to specific plasma proteins’?“, the thyroxine-binding globulin (TBG, an inter a-globulin), pre-albumin (TBPA) and albumin (TBA). The affinities of the proteins for T, and T4 are different. The binding of T3 to TBG is about 4 times weaker as compared with T,, TBPA will only bind T, and the aflinity of TBA is very weak for both hormones. Normally, only 1% of T, and o.I~,~ of ?;I is in the free, biologically active, form. The principle of the resin and Sephadex gel filtration techniques is to measure indirectly the concentration of thyroid hormone by evaluating the saturation state of the serum. To a system such as serum, containing free and bound thyroid hormone CEin.C/&n.Acta,
20 (1968)
rrp-126
114
JACOBS, Z@SI)AG
and partly unoccupied binding proteins, radioactive thyroid hormone (T$ or T,) is added. The radioactive compound will compete with the inactive hormone for the protein-binding sites and will be bound to the free sites. Addition of a resin (or gel fiitration) will, after equilibration, result in a partition of radioactive and inactive hormone between protein and resin (or gel). The amount of resin(gel)-bound radioactive hormone corresponds to the hormone content of the sample. The first test based on this principle, was described by Hamolskys, who used red blood cells instead of a resin. The uptake of i~lS1 I]T, by red cells appeared to be increased in h~~rth~~oidisn~ and reduced in mysedema, compared with values obtained in normal subjects. Since it soon became evident that the red blood cells only act as a non-specific acceptor for the T3 hormone, ion exchange resins were introduced in order to avoid important sources of error {hemolysis, hematocrit corrections, intrinsic erythrocyte abnormalities). Various resins have been studied as to their ability to remove TX from the serum proteins and to differentiate between sera of patients with nnrmo-, hyper- and hypofunction of the thyroid gland”-fi. Complete sets of ready-to-use reagents (T, test kits) are now commercially available from several sources*, as well as a special equipment for the radioactivity measurements involved in the test**. In an isotope laboratory with normal set-up, however, these “facilities” are not needed. In this in~~estigaiion, the resin Amber&e CG 50, type II was used7. Another method fur studying the thyroid hormo~le-binding capacity of serum samples makes use of gel filtration R- 11. Both the molecular sieve effect and the adsorptive properties of the gel play a part in the process of filtration. The proteinbound T, complex cannot enter the gel particles and passes through the bed, appearing in the effluent following an elution volume equal to the void volume of the column. The much smaller molecules of free T, enter the gel particles and can only be eluted from the column with a greater elution volume. MATERIAL
AND
METHODS
was suspended in isotonic saline The resin Amberlite CG-50, type II, 200 mesh*** to a concentration of 40 mg/ml and stored at 5”. The binding capacity of the resin is highest with the freshly prepared suspension and decreases slightly upon storage. If a “reference correction” is applied, as proposed further, the results remain stable and the resin can be stored for z months. Prior to use and during pipetting the suspension must be homogenized by shaking. Tri-iodothyronine
Before use the 1311-labelled 3,5,3’-tri-iodothyronine stock solution (in 5090 propylene glycol, concentration 0.1-0.3 mC 1311per ml, specific activity IO-30 mC per mg of T3t) was diluted in isotonic saline to an activity of O.O+O.I~ &/ml. _.. _-* Triosorb {Abbott Laboratories, North Chicago, Ill.), Triionex (Curtis Nac1ea.r Co., Los Angeles, C&if.). Tresitope (Squibb & Sons, New York}. TBI (Nuclear Consultants Co., St. Louis, &IO.). ** Gammacord (Ames Co., Elkhart, Ind.). Mediac T, counter {Nuclear Chicago Co.. Des Plaiues, Ill.). Thyrodex (Picker Nuclear, White F’tains, N.Y.). * * * I3DH Laboratory Chemicals Division, Poole, England. $ Philips Duphar, Petten, Tbe Netherlands.
THYROID
FUNCTION TEST
115
Decrease in specific activity and release of iodide, which occur during prolonged storage, result in slightly higher resin uptake values (addition of inactive T3 raises the amount of {l”lI]T, bound by the resin, and iodide is strongly absorbed by the resin). Therefore, it is necessary to run a known reference of a plasma pool with each set of determinations, in order to check the behaviour of the tracer and to eliminate further day-to-day variations. The stock solution of [1311]T, can, in our experience, be used 4 to 6 weeks after delivery under these conditions.
Serum was kept at ---20~ prior to investigation. Repeated thawing and freezing had no effect on the resin uptake or gel filtration results. Serum can be stored for six months. Refemnce
plasma pool Human plasma was used as a reference. Transfusion blood was centrifuged, the supernatant plasma decanted and stored at -20~ in IO-ml portions.
Disposable glass tubes Preferably, the tubes should be suited to all steps in the procedure : incubation, centrifugation and counting. Flat-bottomed tablet tubes (length 8.5 cm, outer diameter 1.6 cm) with a polyethylene cap, fitting in the centrifuge as well as in the welltype counter, were selected.
3.5 g of Sephadcx G-25, coarse* is mixed with 20 ml phosphate buffer (0.06 p, according to Stirensen, pH 7.38) and poured into a glass chromatographic column, type 83K, 31rC, with a thermostatic jacket, length 30 cm and interior diameter I cm*:, The column is prepared 1-2 h before use. When out of use, the column can be stored with the Sephadex in the swollen state, after addition of a bacteriostatic agent (0.02’7~ sodium azide).
I ml of diluted [‘“lI]T, solution is pipetted into each of two disposable glass tubes, to serve as a standard of total activity (mean value after subtraction of background activity equals IOO% activity). I ml Amberlite suspension, I ml serum, I ml diluted [1311]Ts solution and 2ml isotonic saline are pipetted into disposable glass tubes, stoppered with a plastic cap and mixed by hand. Next, the tubes are mounted on a stirring wheel, rotating at a speed of zo rev./ min in a thermostat-controlled waterbath at 37”. Since the axis of rotation is placed at a slope of about 45”, this causes the tubes to be turned upside down 20 times every minute, thus ensuring complete mixing of the resin during incubation. The mixture is incubated for a period of 32 min (including 2 min for warming _~.. * Pharmacia Corp., Uppsala, Sweden. ** Pleuger, Amstelveen,
The Netherfands.
C&n. Chim. Acta,
20
(1968)
rrprz(i
up), after which the tubes are centrifuged for IO min at ~naximum speed (~300 XK). The supernatant is discarded carefully and the activity of the resin is counted in a well-type scintillation counter *. After subtraction of the background the uptake is expressed as a percentage of the total activity added (standard rony:;). To correct for slight day-to-day variations in the uptake, a known reference of a plasma pool is run in duplicate with each set of determinations. The fluctuations, mainly caused by aging of the {1311]T, preparation (increase of uptake values) and of the resin suspension (decrease of uptake values), are normally within z’j, of the correct reference value. If more than 520, it seems advisable to renew reagents, although corrections for up to lo’)d were found to give correct results. Correction is carried out as follows: suppose the correct reference v&tie of a plasma pool is ~o.o~$_ If one day a Value Of 3T_3"0 iS 3o.o found, then the results of the unknown samples are multiplied by 3I.3’ I ml serum, I ml diiuted l_KS1X]3’9 solution and I ml phosphate buffer are pipetted, mixed and incubated for 15 min at room temperature. I ml of the mixture is used as a standard (1003; activity) and I ml is carefully pipetted on top of the column and permitted to soak in. The jacket of the column is supplied with water of 37’ from a circulating thermostat, The serum mixture is washed into the column with two further I-ml portions of buffer. Now, ~2 ml of buffer are added ; 2 ml are allowed to flow out and the next 9 ml are collected. The standard is made up to the same volume and both are counted. The activity, in this way eluted from the column, is expressed as a percentage of the standard. Each set of determinations starts and ends with a known reference plasma. The average of this duplicate reference is used as an indicator for the day-to-day fluctuations and to correct the results of the various serum samples. Fluctuations are normally within 27, of the correct reference value and never exceed ;c”,;. Between determinations, the column is washed with 20 ml of plasma and IO 1111 of huffer in order to remove the free T, from the gel and to make the column ready for use for the next filtration. After 6-7 determinations the column should be stirred. About 20 determinations can be done on the same column, without renewing the gel.
After a fasting period of 12 h, a test dose of 10 pC latf (NaW, isotonic solution, specific activity more than 5000 C. 131Xper gram of iodine **) is given orally to the patient. After zq and 48 h the total uptake of the gland is measured by means of a scintillation counter, AaC crystal **t p laced in front of the neck at a distance of 30 cm. In a thyroid phantom (according to the Recommendations of the International Atomic Energy Agency) a standard is placed with an activity equal to the administered dose in a volume of 30 ml and the activity is measured in the fame way as in the patient. This standard serves as a JUO% activity. After subtraction of the background the percentual uptake can be calculated. Normal range in our hands 30-70:/o after zq h ; 48-h value about the same or up to 50/b lower. * l?hilips, type PW 4119/w_ crystal dimeasicrc+: I 7 mm diameter x 35.5 mm. ** Philips Duphar, P&ten. The hTetherlands. *** Philips, type PW 4x19S crystal diameter r.7.j". CEin. Chtm. ,4&a, 20 (1968) 1i3--E26
T.T~* diameters
z* height, well dimensions:
THYROIL)
FUNCTION
To check
117
TEST
the thyroid
uptake
measurements,
the
1311 excretion
in urine
is
estimated simultaneously (sum of 48-h thyroid uptake and 48-h urinary excretion normally represents about 95Oj of the administered dose). Urine is collected over 24 and 48 h separately. IO pC 1311 is made up to I 1 water to serve as a standard. The activity of the urine portions and of the standard is measured by counting 5 ml samples of each, in a scintillation counter, well-type crystal. After subtraction of the background and considering the volume of the 24- and 48-h collections, the percentual excretion
can be calculated.
Clinical material In order to establish
normal
values for resin uptake
and gel filtration,
blood
samples were drawn from 21 donors of the Red Cross Blood Transfusion Service. In the group of patients suspected of thyroid disorders, 49 subjects appeared to have normal thyroid function, 18 had hyperthyroidism and in IO, the thyroid activity was deficient. In nearly all cases the diagnosis was confirmed by [1311]thyroid uptake and protein-bound iodine (PBI)*. Blood samples were taken from 16 normal, pregnant women in the sixth month of pregnancy. These patients all had normal thyroid function. Anothergroupof 16 patients with normal thyroid function but abnormal serum proteins were investigated. They suffered from cirrhosis protein-losing
of the liver (I), Kahler’s
gastroenteropathy
(I), nephrotic
disease (7), paraproteinemia
syndrome
(2) and neoplasm
(4),
(I).
RESULTS Normal values, hyperthyyroidism, hypothyroidism The values for the T, resin uptake in 21 normal subjects
(donors Blood Trans-
fusion Service) ranged from 22.0 to 27.4% (mean 24.3’,&, S.D. 1.3%). The determinations were carried out in duplicate (difference between duplicates less than 17;). The S.D., as calculated from these duplicate determinations, was 0.3%. Of 49 euthyroid patients (Table I), 42 had values within the range of 22 to 28%. Two patients (39 and 43 of Table I) received oral contraceptives. The remaining 5 patients (I, 22, 37, 45 and 46 of Table I) had values between 20.5 and 21.4~&. In 18 hyperthyroid patients (Table II) the T, resin uptake values were in the range of 30.2 to 45.0:/,. In 5 of these patients T, resin uptakes were measured before and after treatment with propylthiouracil. As can be seen from Table III, all values decreased markedly upon treatment. The resin uptakes in g of IO patients with hypothyroidism (Table IV) were below zooA, (17.8 to 19.4%). In the tenth patient a borderline value of 20.6:/, was found. In conclusion it can be said that in patients suspected of thyroid dysfunction, T, resin uptake values above 28.5% are strongly indicative of hyperthyroidism, the range of 28.0 to 28.5% representing borderline cases of doubtful hyperfunction. Furthermore, values below 20.5% strongly support the diagnosis of hypothyroidism, with a borderline range of 20.5 to 21.0%.
* PBI determinations (Auto-Analyzer) were carried out in the Clinical Chemical the “Onze Lime Vrouwe Gasthuis”, Amsterdam (head: Dr Th. Strengers). Clin. Chim.
Acta,
Laboratory
20 (1968)
113-126
of
JACOBS, ZONDAG
118 TABLE
I
INDIVIDUAL VAL.UES FOR CHOLESTEROI., PH. [ 1311]~~~~0~~ UPTAKE, FILTRATION IN CLINICALLY EUTHYROID PATIENTS
No.
I 2
3 4
Cholesterol mg %
303 ‘87 290 267 206
;
13 I4 x5* 16 ‘7 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38* 39 40 4’ 4” 43 44 45 46 47 48 49
%
6.2 8.5 5.7 7.6
2
9 JO II I2
[lslI
PBI M
165 212 206
4.5 10.1 6.7
196 185 215 187 229 270 336 192 265 ‘43 243 230 268 186
5.6 8.2
-
-
-
326 262 I75 232 225 170 265 238 2O4 236 264 198 226 224
21.4
77.6 j4.2
73.1 53.7 40.7 65.1
44.8 65.3 38.7 48.3 71.4 46.5 70.3 55.6 61.7
4.9
4.’ 78.6 58.0
5.4 8.4 6.6 6.0 -
26.1
48.5 65.0
6.6
5.9
IO.4 3.7 6.2 13.1 7.2 4.2 4.0 15.7 8.4 6.8 6.1
uptake
68 h iland
8.4 5.1
4.7 7.0 6.0
Thyroid
24 k gland
55.6 52.5 j9.6 42.5 88.0 44.8 54.2 77.5 59.9 56.8 46.6 48.5 63.4 25.9 43.4 34.5 41.8 59.2 71.2 46.0 18.6 38.5 43.3 52.8 56.3 52.8
346 350 212
6.1 6.0
35.3 29.8
5.2
65.9 52.6
184
6.3
34.3 43.8
44.2 49.9 70.1 45.8 69.3 58.9 64.8 50.5 64.7 I.7 78.2 58.5 56.8 53.1 54.8 43.9 93.6 47.9 33.7 77.6 59.9 55.9 43.3 48.9 49.4 26.7 43.5 36.2 46.2 61.4 64.9 50.0 19.2 40.7 47.7 51.3 51.9 50.0
RESIN
UPTAKE
% 24 h urine
40.5 50.7 25.6 24.5 22.8
48.4 9.5 60.2 57.4 58.9 47.1 40.2 22.6 47.2 73.4 57.4 14.5 56.3 41.5 45.7 58.8
34.9 33.6 56.2 42.8 42.0
40.3 44.8 41.0 27.0
45.5
30.2
T, %
urine
9.3 I.3 3.8 2.5 I.1 8.0 I.9 0.6 I.5 0.7 3.3 2.1 3.2 0.5 16.5 0.3 0.8 I.2 2.L
44.0 43.7 43.8 38.1 4x.7 7.5 34.9 35.6 17.2 35.2 40.6 48.6
Proteinhound
48 k
55.4 31.3 25.9 37.9 37.1 4.9 24.3 3.2 52.3 31.7 19.8
2.7 3.3 0.2 2.3 I.5 I.3 I.2 1.2 I.3 2.7 9.4 1.8 3.2 3.2 1.0 0.1 4.5 11.3 I.4 2.9 0.9 0.6 0.7 2.3 11.7 2.0 1.8 12.6 10.6
AND
20.j
23.5 26.2 26.7 27.5 24.0 26.8 27.0 27.4 23.7 24.4 25.0 23.6 23.6 27.5 26.0 26.2 '3.7 24.1 26.8 23.6 20.5 24.7 23.6 25.0 26.6 26.6 25.4 22.8 27.4 22.1 23.2 27.4 22.6 27.4 26.2 21.4 24.8 19.1 27.9 25.6 23.5 18.6 22.1 20.6 21.0 24.8 22.6 24.8
64.2 55.0 53.8 52.1 55.2 57.6 50.0 54.8 57.8 56.4 59.3 58.8 56.3 52.4 54.0 54.0 56.2 56.7 51.8 -5 8 2 63.4 57.1 58.3 56.3 58.3 53.1 54.6 58.2 51.0 58.0 61.1 70.4 56.2 58.2 52.2 64.2 59.8 67.7 50.7 60.0 60.7 68.0 62.3 6~.4 61.6 60.8 58.4 60.6 -._-
* Contaminated
Clin. Chim.
Acta,
with inorganic or organic iodine.
20 (1968) 113-126
GEL
THYROID
FUNCTION
INDIVtDUAL
VALUES
FILTRATION
IN
TEST
FOR
1x9
CHOLESTEROL,
WYPERTHYROID -“_.”
~__.._._ I
L 3 4 5 6 ii
UPTAKE,
[‘311]THYROIU
uptake __--.48 h gland
24 h gland
__..._--.185 IO.8
24 h urgne
48 h urine I.3 0.6
__ .._~_
72.4 90.5 77.3 59.6 82.5 31.0
IY”3 1x.4 x2.g 4.9 30.9 -1o.4
“_ 42.9 4.5.3 4z.4 48. x
2.0 3.9
35.5 35.9 34.3 31.6 30.2 38.8 34.2
39*4 36.6
r+a
‘84 IT8 ‘3-t
12.9 x3.8 14,s 22.3
Sr.3
77.1
j-8
400-3
40.3
27.3
12.1
35-9 38.0
77.1
73.6 87.2
IO.0 Il.3
0.7 0.5
45-o 33-7
27.7 42.6
72.2 49.7
4.2 I‘+“6 6.4 _.~”
4.2 7.8
38.4 35-7 43.1 34.8 35.3
30.4 45.1 39.fi 42.0
35.0 3r.4
42.3 47.7
187 193
24.0
89.9
I2 13 ‘4 15 16
226 1.36
12.0
78.9 71.3 -
9~4
27.6 .“._ 14.1
-
RESIN UPTAKE
28.8
--
10.2 1.4 0.5 ._. ---_. . with inorganic or organic iodine.
‘7 ‘54 IX* 219 ____..__~ * Contaminated
AND
GEL
FILTRATIOA
IN HYPERTHYROID
PROPYI.THLOlJRACIL
x0.
Beforc
2.8
-..
77*” _
IS.2
PATIENTS
BEFORE
treatment Protein-bouvzd
%
T, “,‘o
.
A fteer treatment L_--.___.. Resin @take 0’ ,I”
T, “/b
_--
20.0
60.5 68.5
3 4
24.2 22.7
55.9 -
5
XL%
39.4
20.2
62.9
2
4sj.O
40.2
AFTER
TREATMEX
T
Protein-bounh
30-4 3c.4 42-4 42.3
I
AND
30.7
-l_._ll_________
-P??B uptake I~.
I.2
3.0 o.h
38.8 38.4 31.6 35.0
21.5
-
IV
INDIVII)UAL VAl_‘UBS IN
FfLTRATiON
FOR
NYPOTWYROID
CHOLESTEROL,
FBI,
PATIENTS
[=l~]THYROID
Ckd-
PBI
[1311] Thyroid
ester01
@“&%
-__
mg % ----^
24 k gland
I 2 3 4 5 6
542 467 447 500 258 423
I.3 x.9 %“7 1.t I.0 a.8
7.8 11.7 6.3
206 325
L.3 “.5
10.6
rag
s9.5
-_
1.1
14.7
IS.+
322
-_
49.0 60.7
-
@take ‘7; --1--_-_ 48 J? 24 k wine gland “_. 6.9
I.0
S-5
___~
UPTAKE,
__.-I___
ivo.
B
.-
156
10 II
GEL
“/s
16.5 ix.4
20.0
I0
AND
115 I28
152
s7
UPTAKE
79.6 87.3 82.5 44.9 62.4 84.8 42.1
9
TABLE
RESIN
.~~-^-.“l.l..
[1311]Thyroid
No.
WITH
FBI,
PATIENTS
6O._y
II.8
59.4
5.2 -~ 0.42
55.4
5-5
I-._ 67.0
_._~
RESIN
UPTAKE
X?\‘D GEL
____l_---.
48k r, urzne
Resira uptake
__x____
Proteinbound
”/0
T, %
18.3 18.3 19.0 18.6 17.8 20.6
67.8 66.1 66.0 68.5 67.0 60.4
r3.9
19.2 18.g
67.5 67.g
$5.6 20.4
x9.4 x9.1
66.9 -._ . . _~
14.6 12.9 21.0 x9.6 -
Clin. Claim. Acfa,
20 (rg68)
rr3--126
JACOBS,
ZONIIAG
In 16 normal pregnant women the T, resin uptake values ranged from 15.1 to 17.674 (mean 16.4, SD. 0.8). In a patient whose pregnancy terminated in still-birth, a value of (16.5
20.8
was found. In 4 women receiving as can be seen in Table V.
the “pill”, the uptakeswere
low also
to 19,57/o),
TABLE
\i
ANI) GEL FILTRATION IN EUTHYROID AND W,X’ERTHYROID WOMEN DrRlSC ADMINISTRATION OF ORAI. CONTRACEPTIVES (LYNDIOL 2.5)
RESIN
UPTAKE
X0.
Cow.ditinn
lie.% “’ 0
I
euthyroid euthyroid euthyroid euthyroid
19. I 18.6 16.5
67.7
19.5
07.0
hyperthyroid hyperthyroid hyperthyroid
25.2 24.0
2 3 4 5 0
7
uptnire
Prcfsisz-bowzd 2’ 0 3 ,I> 08.0 _
00.9
27.j
serum @rote&s In patients with marked abnormalities in serum protein pattern the T, resin uptake test can give false results, with values suggesting hyperthyroidism in cases with normal thyroid function. It seems obvious that decreased levels of qlobulins, pre-albumin and albumin can cause increased resin uptake. From our material
Ab~wmal
(Table VI), this is indeed likely, although some exceptions (9, 16 of Table VI) occur. The increased uptake value of patient r3a was probably due to the very low serum albumin value: with increasing albumin concentration, the resin uptake decreased (13b). IIowever,
the limited data presented
sions concerning
this problem.
TAULE
here do not allow of any definite conclu-
VI
RESIP; UPTAKE VALUES IN EUTHYROID PATIEXTS WITH ABNORMAL SERUM PROTEIN
II.60
8.60 9.45 5.70 10.00 7.45 6.00 7.40 6.10 7.90 IO.50 5.80 4.80 4.30 3.90 3.60 4.90
c+ A- az-
g 0’ ;o
Globrrlil?~ g y* __-.. . .___
1 5s .?.IS 3.10
0‘45
0.30 0.15
3.45 3.85 3.35 3.40 3.60 3.05 2.90
0.q
3.85 3.85 0.50
0.25
x.25 0.75 0.6j 0.80 0.80
0.95 0.85 I.60 I.00
I.55 x.75 2.05
0.80
“-45
r.os
PATTERN
Diagnosis
Albumin
i.ZO
-.
Cirrhosis of the liver IgA-Kahkr IgA-Kahler IgA-Kahler IgG-Kahler IgG-Kahler IgA-paraproteinemia IgG-paraproteinemia lgG-paraproteinemia IgG-paraproteinemia Kahler Plasmocytoma Nephrotic syndrome Nephrotic syndrome Nephrotic syndrome Gastroenteropathy Generalized carcinomr
3 3 ..5 JO.5 40.9 31.1 28.7 2x.2 Xj.6 28.5
32.3 20.6 23.3 33.7 21.3 28.4 30.0 32.3
THYROID
FUNCTION
121
TEST
Sephadex gel filtration Most serum samples tested with the T, resin uptake test were investigated the Sephadex gel filtration technique as well (Tables I-VI). Most determinations carried out in duplicate. The SD., as calculated from duplicate determinations, 1.5%. Differences of up to 5% occurred, but were rare.
with were was
Normal values with this technique were in the range of 50 to 650/b. Of 48 euthyroid patients (Table I) 45 had values within this range, two receiving oral contraceptives were accordingly high (67.7?; and 68.0%) and theremaining patient (33 of Table I) had an unexplained high value of 70.4:/~. In hyperthyroidism (Table II) values of 27 to 48% were found, less than 5094 indicative of hyperfunction. Of the IO hypothyroid patients (Table IV), o had values between 66.0 and 68.5%. Patient 6, the one with a borderline resin uptake, had a gel filtration value of 60.4O,:,. In general, values above 659/o can be considered indicative of hypothyroidism. However, in pregnancy, high results are found as well (70 to 76:/,, mean 73.394,
S.D. 1.8:‘,)
Exfierimental The amount mental conditions,
and the same holds true for women taking the “pill” (Table V).
;1311]T, absorbed by the resin is dependent on several experiof L including temperature and time of incubation, homogenization
during
incubation, pH and buffer molarity of the mixture, amount of resin and with amount of inactive T3 (or T4) addede. In our experience and in accordance other+7, the uptake of T, by the resin increases with increasing temperature. If
incubation is carried out at room temperature in combination with shaking, uptake values are much lower and the difference between the highest and lowest values is only about half of that obtained at 37O. Incubation at 37O but without shaking, results in lower values as well (normal range about 15 to 20%) ; besides, the reproducibility appears to be rather poor in comparison with the proposed method. Required reproducibility
and optimal separation into the three clinically important classes was shaking during incubation. achieved at a temperature of 37O, under continuous Incubation during I h instead of 30 min or increasing the amount of resin per test, raises the resin uptake without improvement of the separation. The same holds true for addition of inactive T,. Most methods for the T, resin uptake include cumbersome washing of the resin. Repeated washings with isotonic saline (each time followed by centrifugation), up to 5 times in succession, are recommended. Apart from being time-consuming, washing involves the risk of loss of activity from the resin or loss of the resin itself. Therefore, attempts were made to avoid washing. From experiments made for this purpose it appeared that the remaining activity of the resin is less after each of the first 3 or 4 washings, only reaching a more or less constant level after some 4 to 6 washings. However, this was somewhat precarious. In a further study, including samples of normo-, hyper- and hypothyroid subjects, a number of estimations were carried out first without washing and then after three washings. In this way it could be shown that washing is not necessary and has no advantage. After three washings, the measured activities were found z to 30/o lower over the whole range of low, normal and high values, so that washing had no influence on the measure of separation. Clin. Chim.
Acta,
20 (1~68)
113-126
122
JACOBS, ZONDAG
The Sephadex gel filtration is based on the fact that protein-bound jrslI]TZ will pass relatively rapidly through the gel bed, unlike the free /131IjT,, which can only be &ted from the column with the aid of fluids having a strong affinity for T,, such as serum or plasma. The advantage of this column chromatographic technique over the resin uptake methods lies in the possibility of fractionation by means of separate collection of the effluent. Thus, in the case of contamination with radioactive iodide (as in samples from patients with previous administration of Nal”lI, or as in case the jlBfI]Ta preparation contains free radioactive iodide), it becomes possible to collect the [13r1]Ts from the column without elution of the more firmly bound iodide. However, in all our experiments, there appeared only one peak of activity in the effluent when eluting the column with buffer, representing the activity of the .1311]TZ bound by protein. So far as our !/ 1311]Ts preparation was contaminaied with iodide, this did not give a separate peak of activity. If, after elution with buffer, the remaining activity of the column was eluted with plasma, the recovery of the total activity applied to the column was in the range of 95.0 to 97.37;. The gel filtration technique was carried out roughly
according
to Cuar6n8.
In
preliminary experiments with fractionated collection of the effluent in I-ml portions, it appeared that the peak of activity is mainly obtained in the portions 3, L+,5 and 6. The first 2 ml running from the column contain no activity and can therefore be discarded. Routinely, the next 9 ml were collected and measured, representing the amount of [l”iI]T, bound by the serum proteins. The activity in the rest of the effluent was very low and can be neglected. Washing with 20 ml plasma was shown to be completely effective in removing the free /1311!T, from the column. Coarse-grained Sephadex was chosen, which has the advantage over the medium-grade quality of greater effluent flow (about z ml/min). The gel filtration process is rather temperature-sensitive.
With increasing
gel. In order to avoid inconstant a constant column temperature water jacket.
Pre-incubation
out at room temperature Some experiments
temperature
more T, is bound by the
results caused by fluctuations in room temperature of 37” was realized, using columns provided with a
of the serum samples with T, can equally well be carried
as at 37’; this does not make any difference. were carried out with the method described
by
Hvid
Hansen”“. This method can be considered as a resin uptake method, except that the resin is substituted by Sephadex. However, in our hands this technique was unsuccessful. After each washing, the remaining activity of the Sephadex was found less, even after 0 or 7 washings, without reaching a constant level. Reproducibilitywas less satisfactory and the uptake range was not sufficient for accurate measurements (below IO:,
of added activity).
COMPARISON
WITH OTHER METHODS AND DISCUSSION
Many thyroid function tests are now available for clinical practice. Since each test has its own advantages and pitfalls 13, it is not advisable to rely on a single procedure in thyroid testing. On the contrary, it can be stated that several different tests are needed in order to attain sufficient diagnostic precision. The [*311]thvroid uptake test is unique in so far as it gives a direct measure of thyroid activity ii U&IO.unfortunately, a number of extra-thyroid factors affect the results and there is a rather large overlap in physiological and pathological values,
which often makes their interpretation more or less uncertain. For example, for the 24-h thyroid uptake, a wide range of 30 to 70% was somewhat arbitrarily accepted as normal in our laboratory. Nevertheless, a 24-h uptake of over 70% can be found in euthyroid subjects with iodide deficiency (probably the case in patients 2,8,16,22, 25 and 36 in Table I), whereas values below 30% are encountered in normal patients patients I, 15 and 38 in Table I, and with iodide excess (iodide “contamination”; patient 18 in Table II). Normal values may be established at any time between 2 and 48 hours after administration of the tracer dose, and measurement at two or more different times gives more information, especially in view of the rapidity of 131f turnover. Routinely, the 24-and 48-h uptakes were measured in our laboratory, but in some instances a 6-h value is required as well, to establish the diagnosis of hyperthyroid&m. If an accelerated uptake is accompanied by rapid conversion of the lslI into the hormone form by the thyroid gland, then the maximum uptake value can be attained well within 24 h after 13iI administration. This can be illustrated by patient 4 of Table II : 6, 12,24,48 and 72-h thyroid uptakes, respectively, were 80.5%, 59.3%, 44.9%, 28.8% and 23.1%. The normal 24-h uptake values in the hyperthyroid patients 5,7 and g of Table II could be explained in the same way. The widely used determination of PI31 is generally considered to represent one of the most reliable indices of the state of thyroid function. We therefore compared the resin uptake test with the corresponding PI31 determinations. The results are shown in Fig. r. As can be seen from this figure, the correlation is good. A number of patients need further comment. Two subjects (8 and 30, Table I) had resin uptake values in the upper normal range, whereas the PBI of both serum samples was elevated, Clinically, it was doubtful whether they were euthyroid or hyperthyroid. Tn a third patient (33 of Table I) the elevated PBI was probably due to iodine contamination, as this patient was undoubtedly euthyroid, in accordance with the resin . ..m.2%6
Fig.
I.
Relationship between resin uptake and PBI, x = Contaminated with iodine.
r24
7(
JACOBS,
5.
A* ,
ZONDAG
‘*
.
5( 3a’ c z 0‘ J( )11 .t i 2 El a l(
Fig. 2. iodine.
Relationship
between gel filtration (protein bound TJ and PRI.
x = ~~~t~rn~nated with
uptake value (the gel ~ltration value of this patient, in the hypotl~yroid range, will be discussed later). The borderline resin uptake value of 20.6(~;~in a clinically distinctly hypoth~oid patient (No. 6 of Table IV) was somewhat too high in comparison with the very low PBT. Finally, in two patients clinically classified as normal (22 and 45 of Table I), a normal PBI was accompanied by low borderline resin uptakes of 20.5% and 20.6%. At least in one of them (No. 45) the assumption of a slight hypofunction seems justified, considering the rather high cholesterol level and the low [1*ll]thyroid uptake ipzvitro. As can further be seen from Fig. I, the resin uptake test permits the correct classification of patients in spite of iodine contamination. In Fig. 2, showing the ~ornpa~so~ between gel filtration results and PBX, nearly the same picture is seen, Since the correlation between resin uptake and gel filtration (Fig. 3) is very good, the resemblance between Figs. I and z is not surprising and requires no further ~o~l~lent. There is only one exception to be made. Patient 33, Table I, clinically euthyroid, presented a completely incomprehensil)le set of figures: high PBI (probably due to iodine contamination), normal resin uptake and hypothyroid gel filtration value. No explanation was found for this. Fig. 4 shows a summary of the resin uptake results in connection with the clinical classification of the patients. The separation between euthyroid and hyperthyroid patients is excellent; between euthyroid and hypothyroid patients the separation is probably slightly less sharp. Resin uptake values are always very low during normal pregnancy. As such, the test can be used as a kind of pregnancy test and can even play a part in predicting an abortion. In one patient, intrauterine foetal death had probably occurred. Three weeks later the resin uptake was zo.S%, supporting the diagnosis. The pregnancy terminated in St&birth. During the use of oral contraceptives, the values for resin uptake are also low. Clin. C?&. A&, 20 (18&H)I I.$-126
THYROID
FUNCTION
f;ig. 3. Relationship with iodine.
TEST
between gel filtration [protein bound T.J and resin uptake. x = C~nt~~ina~ed
If, on the other hand, resin uptake is normal in a patient taking the pill, this suggests ~lyperthyroidism~ as could be established in 3 patients (see also Table V). Finafly, it must be borne in mind that, in patients with grossly abnormal serum protein pattern, resin uptake values in the hyperthyroid range can be found which are not due to abnormal thyroid function, as could be demonstrated in IO out of 16 patients with dysproteinacmia. Fig. 5, summarizing the gel filtration resuIts
l
hype&h.
euth.
Fig. 4. Scatter diagram terine foetal death.
hypoth.
F-w
pill euth.
II hyperth.
disprot.
of resin uptake resutts in various clinical conditi~lls. Open circle I intrau-
.o8:.
d:
. --
:it :*
.A:.. .:jff:
l ..
.
0
~
I-
: . .. .*. .O-
1.
l
l
0
!
*
‘
hj/pert h.
cut h.
hypoth.
p=gn.
I PiII
euth.
Fig. 5. Scatter diagram of gel filtration results in various clinical conditions. terine foetal death.
Open circle = intrau-
in connection with the clinical classification, shows the same picture as Fig. 4 and leads to the same comments. In conclusion, the resin uptake test in the proposed modification has proved to be accurate, and particularly useful for the rapid screening of thyroid dysfunction. Although it is slightly more time-consuming, the same holds true for the Sephadex gel filtration technique. The advantage of both tests over PBI and [1311]thyroid uptake is vitro is that previous ingestion of iodine has no influence on the results. Furthermore, the resin uptake test does not require the administration of radioactive iodine to the patient. Tests can therefore be repeated at short intervals, and thus be of special value in following the effect of treatment. ACKNOWLEDGEMENTS We wish to express our gratitude to the Staffs of the Departments of Internal Diseases of the St. Elisabeth’s Hospital and the St. Joannes de Deo Hospital, Haarlem, for their cooperation in providing the clinical data of the patients. We also thank Dr. L. van Beugen for reading the manuscript and his helpful criticism and Miss A. Kruyt for her technical assistence. REFERENCES
1 J. ROBBINS AND 1. E. RX=, PhysioE. Rev., 40 (1960) 415. 2 S. H. INGBAR, J. C&n. Invest., 4~ (x963) 143. 3 M. W. HAMOLSKY, M. STEIN AND A. S. FXEEDBERG, I. CZin. Endocrinol. Metab., 17 (1957) 3.3. 4 M. L. MITCHELL, A. B. HARDEN AND M. E. O’ROURKE, 1. Clin. Endocrirzol. Mefab., 20 (1960) 5 G 7 8 ‘9 10
1474. Ii. STERLING AND M. TABACHNICK, J. Clin. Endocrinol. Metab., 21 (1961) 456. VI’. BBRNER, M. HOHBACH, E. MOLL AND H. BAYER, Klin. Wochschr., 44 (1966) 973. M. G. WOLDRING, A. BAKKER AND H. DOORENBOS, Acta Endocrinol., 37 (rg6r) 607. A. CV~R~N AND M. E. FUCUGAUCHI, Acta Endocrinol., 46 (1964) 161. I?. C.. SCRIBA, R. LANDGRAF, H. G. HEINZE AND K. SCHWARZ, Klin. Wochschr., 44 (1966) 69. I’. C. SCRIRA, H. G. HEINZE, R. LAXDGRAF, K. W. FREY AND K. SCHWARZ, Klin. Wochschr.,
xx
44 (1966) 131. R. GRKNICHER, A. BURGER, E. GFELLER AND H. STUDER, 2. Klin.
(1967) 1x7. IZ H. HSID HANSEN, Scaad. .J_ C&in. Lab. 13 J. C. Srsso~, J. Nzsclear Med., 6 (1965) Clin.
Chim.
Acta,
20 (1968)
rr3-126
Invest., 853.
18 (x966)
240.
Chem.
Klin.
&o&em.,
5
CLINICA CHIMICA ACTA
SIMPLE
SCREENING
TEST
FOR THE
EVALUATION
OF THYROID
FUNCTION (EXPERIENCE BASED
WITH
ON RESIN
TWO IN-VITRO
UPTAKE
;1311]THYROID
AND SEPHADEX
FUNCTION
TESTS
GEL FILTRATION)
SUMMARY
A modification of the [1311]tri-iodothyronine (TJ resin uptake test was investigated. A simple, fast and convenient method was elaborated, eliminating several time-consuming steps in the procedure, such as pre-incubation of the sample, preweighing of resin, cumbersome washing procedure, furthermore choosing convenient temperature conditions (handling at room temperature, incubation at 37’) and limiting the incubation time and the number of radioactivity measurements needed. The test can be carried out in a routine isotope laboratory, without special equipment. The results of the test were compared with a Sephadex gel filtration T8 test and, in the case of suspected thyroid disorders, also with the protein-bound iodine (PBI) and the [1311]thyroid uptake test in vitro. With each test (resin uptake and gel filtration), most patients can be classified correctly as euthyroid, hyperthyroid and hypothyroid ones, according to the clinical diagnosis. However, values in the hypothyroid range are found during pregnancy and under the influence of oral contraceptives, whereas gross abnormalities in serum protein pattern can give rise to values in the hyperthyroid range.
INTRODUCTION
After secretion into the blood, the thyroid hormones thyroxine (TJ and tri-iodothyronine (T,) are, for the most part, bound to specific plasma proteins’?“, the thyroxine-binding globulin (TBG, an inter a-globulin), pre-albumin (TBPA) and albumin (TBA). The affinities of the proteins for T, and T4 are different. The binding of T3 to TBG is about 4 times weaker as compared with T,, TBPA will only bind T, and the aflinity of TBA is very weak for both hormones. Normally, only 1% of T, and o.I~,~ of ?;I is in the free, biologically active, form. The principle of the resin and Sephadex gel filtration techniques is to measure indirectly the concentration of thyroid hormone by evaluating the saturation state of the serum. To a system such as serum, containing free and bound thyroid hormone CEin.C/&n.Acta,
20 (1968)
rrp-126
114
JACOBS, Z@SI)AG
and partly unoccupied binding proteins, radioactive thyroid hormone (T$ or T,) is added. The radioactive compound will compete with the inactive hormone for the protein-binding sites and will be bound to the free sites. Addition of a resin (or gel fiitration) will, after equilibration, result in a partition of radioactive and inactive hormone between protein and resin (or gel). The amount of resin(gel)-bound radioactive hormone corresponds to the hormone content of the sample. The first test based on this principle, was described by Hamolskys, who used red blood cells instead of a resin. The uptake of i~lS1 I]T, by red cells appeared to be increased in h~~rth~~oidisn~ and reduced in mysedema, compared with values obtained in normal subjects. Since it soon became evident that the red blood cells only act as a non-specific acceptor for the T3 hormone, ion exchange resins were introduced in order to avoid important sources of error {hemolysis, hematocrit corrections, intrinsic erythrocyte abnormalities). Various resins have been studied as to their ability to remove TX from the serum proteins and to differentiate between sera of patients with nnrmo-, hyper- and hypofunction of the thyroid gland”-fi. Complete sets of ready-to-use reagents (T, test kits) are now commercially available from several sources*, as well as a special equipment for the radioactivity measurements involved in the test**. In an isotope laboratory with normal set-up, however, these “facilities” are not needed. In this in~~estigaiion, the resin Amber&e CG 50, type II was used7. Another method fur studying the thyroid hormo~le-binding capacity of serum samples makes use of gel filtration R- 11. Both the molecular sieve effect and the adsorptive properties of the gel play a part in the process of filtration. The proteinbound T, complex cannot enter the gel particles and passes through the bed, appearing in the effluent following an elution volume equal to the void volume of the column. The much smaller molecules of free T, enter the gel particles and can only be eluted from the column with a greater elution volume. MATERIAL
AND
METHODS
was suspended in isotonic saline The resin Amberlite CG-50, type II, 200 mesh*** to a concentration of 40 mg/ml and stored at 5”. The binding capacity of the resin is highest with the freshly prepared suspension and decreases slightly upon storage. If a “reference correction” is applied, as proposed further, the results remain stable and the resin can be stored for z months. Prior to use and during pipetting the suspension must be homogenized by shaking. Tri-iodothyronine
Before use the 1311-labelled 3,5,3’-tri-iodothyronine stock solution (in 5090 propylene glycol, concentration 0.1-0.3 mC 1311per ml, specific activity IO-30 mC per mg of T3t) was diluted in isotonic saline to an activity of O.O+O.I~ &/ml. _.. _-* Triosorb {Abbott Laboratories, North Chicago, Ill.), Triionex (Curtis Nac1ea.r Co., Los Angeles, C&if.). Tresitope (Squibb & Sons, New York}. TBI (Nuclear Consultants Co., St. Louis, &IO.). ** Gammacord (Ames Co., Elkhart, Ind.). Mediac T, counter {Nuclear Chicago Co.. Des Plaiues, Ill.). Thyrodex (Picker Nuclear, White F’tains, N.Y.). * * * I3DH Laboratory Chemicals Division, Poole, England. $ Philips Duphar, Petten, Tbe Netherlands.
THYROID
FUNCTION TEST
115
Decrease in specific activity and release of iodide, which occur during prolonged storage, result in slightly higher resin uptake values (addition of inactive T3 raises the amount of {l”lI]T, bound by the resin, and iodide is strongly absorbed by the resin). Therefore, it is necessary to run a known reference of a plasma pool with each set of determinations, in order to check the behaviour of the tracer and to eliminate further day-to-day variations. The stock solution of [1311]T, can, in our experience, be used 4 to 6 weeks after delivery under these conditions.
Serum was kept at ---20~ prior to investigation. Repeated thawing and freezing had no effect on the resin uptake or gel filtration results. Serum can be stored for six months. Refemnce
plasma pool Human plasma was used as a reference. Transfusion blood was centrifuged, the supernatant plasma decanted and stored at -20~ in IO-ml portions.
Disposable glass tubes Preferably, the tubes should be suited to all steps in the procedure : incubation, centrifugation and counting. Flat-bottomed tablet tubes (length 8.5 cm, outer diameter 1.6 cm) with a polyethylene cap, fitting in the centrifuge as well as in the welltype counter, were selected.
3.5 g of Sephadcx G-25, coarse* is mixed with 20 ml phosphate buffer (0.06 p, according to Stirensen, pH 7.38) and poured into a glass chromatographic column, type 83K, 31rC, with a thermostatic jacket, length 30 cm and interior diameter I cm*:, The column is prepared 1-2 h before use. When out of use, the column can be stored with the Sephadex in the swollen state, after addition of a bacteriostatic agent (0.02’7~ sodium azide).
I ml of diluted [‘“lI]T, solution is pipetted into each of two disposable glass tubes, to serve as a standard of total activity (mean value after subtraction of background activity equals IOO% activity). I ml Amberlite suspension, I ml serum, I ml diluted [1311]Ts solution and 2ml isotonic saline are pipetted into disposable glass tubes, stoppered with a plastic cap and mixed by hand. Next, the tubes are mounted on a stirring wheel, rotating at a speed of zo rev./ min in a thermostat-controlled waterbath at 37”. Since the axis of rotation is placed at a slope of about 45”, this causes the tubes to be turned upside down 20 times every minute, thus ensuring complete mixing of the resin during incubation. The mixture is incubated for a period of 32 min (including 2 min for warming _~.. * Pharmacia Corp., Uppsala, Sweden. ** Pleuger, Amstelveen,
The Netherfands.
C&n. Chim. Acta,
20
(1968)
rrprz(i
up), after which the tubes are centrifuged for IO min at ~naximum speed (~300 XK). The supernatant is discarded carefully and the activity of the resin is counted in a well-type scintillation counter *. After subtraction of the background the uptake is expressed as a percentage of the total activity added (standard rony:;). To correct for slight day-to-day variations in the uptake, a known reference of a plasma pool is run in duplicate with each set of determinations. The fluctuations, mainly caused by aging of the {1311]T, preparation (increase of uptake values) and of the resin suspension (decrease of uptake values), are normally within z’j, of the correct reference value. If more than 520, it seems advisable to renew reagents, although corrections for up to lo’)d were found to give correct results. Correction is carried out as follows: suppose the correct reference v&tie of a plasma pool is ~o.o~$_ If one day a Value Of 3T_3"0 iS 3o.o found, then the results of the unknown samples are multiplied by 3I.3’ I ml serum, I ml diiuted l_KS1X]3’9 solution and I ml phosphate buffer are pipetted, mixed and incubated for 15 min at room temperature. I ml of the mixture is used as a standard (1003; activity) and I ml is carefully pipetted on top of the column and permitted to soak in. The jacket of the column is supplied with water of 37’ from a circulating thermostat, The serum mixture is washed into the column with two further I-ml portions of buffer. Now, ~2 ml of buffer are added ; 2 ml are allowed to flow out and the next 9 ml are collected. The standard is made up to the same volume and both are counted. The activity, in this way eluted from the column, is expressed as a percentage of the standard. Each set of determinations starts and ends with a known reference plasma. The average of this duplicate reference is used as an indicator for the day-to-day fluctuations and to correct the results of the various serum samples. Fluctuations are normally within 27, of the correct reference value and never exceed ;c”,;. Between determinations, the column is washed with 20 ml of plasma and IO 1111 of huffer in order to remove the free T, from the gel and to make the column ready for use for the next filtration. After 6-7 determinations the column should be stirred. About 20 determinations can be done on the same column, without renewing the gel.
After a fasting period of 12 h, a test dose of 10 pC latf (NaW, isotonic solution, specific activity more than 5000 C. 131Xper gram of iodine **) is given orally to the patient. After zq and 48 h the total uptake of the gland is measured by means of a scintillation counter, AaC crystal **t p laced in front of the neck at a distance of 30 cm. In a thyroid phantom (according to the Recommendations of the International Atomic Energy Agency) a standard is placed with an activity equal to the administered dose in a volume of 30 ml and the activity is measured in the fame way as in the patient. This standard serves as a JUO% activity. After subtraction of the background the percentual uptake can be calculated. Normal range in our hands 30-70:/o after zq h ; 48-h value about the same or up to 50/b lower. * l?hilips, type PW 4119/w_ crystal dimeasicrc+: I 7 mm diameter x 35.5 mm. ** Philips Duphar, P&ten. The hTetherlands. *** Philips, type PW 4x19S crystal diameter r.7.j". CEin. Chtm. ,4&a, 20 (1968) 1i3--E26
T.T~* diameters
z* height, well dimensions:
THYROIL)
FUNCTION
To check
117
TEST
the thyroid
uptake
measurements,
the
1311 excretion
in urine
is
estimated simultaneously (sum of 48-h thyroid uptake and 48-h urinary excretion normally represents about 95Oj of the administered dose). Urine is collected over 24 and 48 h separately. IO pC 1311 is made up to I 1 water to serve as a standard. The activity of the urine portions and of the standard is measured by counting 5 ml samples of each, in a scintillation counter, well-type crystal. After subtraction of the background and considering the volume of the 24- and 48-h collections, the percentual excretion
can be calculated.
Clinical material In order to establish
normal
values for resin uptake
and gel filtration,
blood
samples were drawn from 21 donors of the Red Cross Blood Transfusion Service. In the group of patients suspected of thyroid disorders, 49 subjects appeared to have normal thyroid function, 18 had hyperthyroidism and in IO, the thyroid activity was deficient. In nearly all cases the diagnosis was confirmed by [1311]thyroid uptake and protein-bound iodine (PBI)*. Blood samples were taken from 16 normal, pregnant women in the sixth month of pregnancy. These patients all had normal thyroid function. Anothergroupof 16 patients with normal thyroid function but abnormal serum proteins were investigated. They suffered from cirrhosis protein-losing
of the liver (I), Kahler’s
gastroenteropathy
(I), nephrotic
disease (7), paraproteinemia
syndrome
(2) and neoplasm
(4),
(I).
RESULTS Normal values, hyperthyyroidism, hypothyroidism The values for the T, resin uptake in 21 normal subjects
(donors Blood Trans-
fusion Service) ranged from 22.0 to 27.4% (mean 24.3’,&, S.D. 1.3%). The determinations were carried out in duplicate (difference between duplicates less than 17;). The S.D., as calculated from these duplicate determinations, was 0.3%. Of 49 euthyroid patients (Table I), 42 had values within the range of 22 to 28%. Two patients (39 and 43 of Table I) received oral contraceptives. The remaining 5 patients (I, 22, 37, 45 and 46 of Table I) had values between 20.5 and 21.4~&. In 18 hyperthyroid patients (Table II) the T, resin uptake values were in the range of 30.2 to 45.0:/,. In 5 of these patients T, resin uptakes were measured before and after treatment with propylthiouracil. As can be seen from Table III, all values decreased markedly upon treatment. The resin uptakes in g of IO patients with hypothyroidism (Table IV) were below zooA, (17.8 to 19.4%). In the tenth patient a borderline value of 20.6:/, was found. In conclusion it can be said that in patients suspected of thyroid dysfunction, T, resin uptake values above 28.5% are strongly indicative of hyperthyroidism, the range of 28.0 to 28.5% representing borderline cases of doubtful hyperfunction. Furthermore, values below 20.5% strongly support the diagnosis of hypothyroidism, with a borderline range of 20.5 to 21.0%.
* PBI determinations (Auto-Analyzer) were carried out in the Clinical Chemical the “Onze Lime Vrouwe Gasthuis”, Amsterdam (head: Dr Th. Strengers). Clin. Chim.
Acta,
Laboratory
20 (1968)
113-126
of
JACOBS, ZONDAG
118 TABLE
I
INDIVIDUAL VAL.UES FOR CHOLESTEROI., PH. [ 1311]~~~~0~~ UPTAKE, FILTRATION IN CLINICALLY EUTHYROID PATIENTS
No.
I 2
3 4
Cholesterol mg %
303 ‘87 290 267 206
;
13 I4 x5* 16 ‘7 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38* 39 40 4’ 4” 43 44 45 46 47 48 49
%
6.2 8.5 5.7 7.6
2
9 JO II I2
[lslI
PBI M
165 212 206
4.5 10.1 6.7
196 185 215 187 229 270 336 192 265 ‘43 243 230 268 186
5.6 8.2
-
-
-
326 262 I75 232 225 170 265 238 2O4 236 264 198 226 224
21.4
77.6 j4.2
73.1 53.7 40.7 65.1
44.8 65.3 38.7 48.3 71.4 46.5 70.3 55.6 61.7
4.9
4.’ 78.6 58.0
5.4 8.4 6.6 6.0 -
26.1
48.5 65.0
6.6
5.9
IO.4 3.7 6.2 13.1 7.2 4.2 4.0 15.7 8.4 6.8 6.1
uptake
68 h iland
8.4 5.1
4.7 7.0 6.0
Thyroid
24 k gland
55.6 52.5 j9.6 42.5 88.0 44.8 54.2 77.5 59.9 56.8 46.6 48.5 63.4 25.9 43.4 34.5 41.8 59.2 71.2 46.0 18.6 38.5 43.3 52.8 56.3 52.8
346 350 212
6.1 6.0
35.3 29.8
5.2
65.9 52.6
184
6.3
34.3 43.8
44.2 49.9 70.1 45.8 69.3 58.9 64.8 50.5 64.7 I.7 78.2 58.5 56.8 53.1 54.8 43.9 93.6 47.9 33.7 77.6 59.9 55.9 43.3 48.9 49.4 26.7 43.5 36.2 46.2 61.4 64.9 50.0 19.2 40.7 47.7 51.3 51.9 50.0
RESIN
UPTAKE
% 24 h urine
40.5 50.7 25.6 24.5 22.8
48.4 9.5 60.2 57.4 58.9 47.1 40.2 22.6 47.2 73.4 57.4 14.5 56.3 41.5 45.7 58.8
34.9 33.6 56.2 42.8 42.0
40.3 44.8 41.0 27.0
45.5
30.2
T, %
urine
9.3 I.3 3.8 2.5 I.1 8.0 I.9 0.6 I.5 0.7 3.3 2.1 3.2 0.5 16.5 0.3 0.8 I.2 2.L
44.0 43.7 43.8 38.1 4x.7 7.5 34.9 35.6 17.2 35.2 40.6 48.6
Proteinhound
48 k
55.4 31.3 25.9 37.9 37.1 4.9 24.3 3.2 52.3 31.7 19.8
2.7 3.3 0.2 2.3 I.5 I.3 I.2 1.2 I.3 2.7 9.4 1.8 3.2 3.2 1.0 0.1 4.5 11.3 I.4 2.9 0.9 0.6 0.7 2.3 11.7 2.0 1.8 12.6 10.6
AND
20.j
23.5 26.2 26.7 27.5 24.0 26.8 27.0 27.4 23.7 24.4 25.0 23.6 23.6 27.5 26.0 26.2 '3.7 24.1 26.8 23.6 20.5 24.7 23.6 25.0 26.6 26.6 25.4 22.8 27.4 22.1 23.2 27.4 22.6 27.4 26.2 21.4 24.8 19.1 27.9 25.6 23.5 18.6 22.1 20.6 21.0 24.8 22.6 24.8
64.2 55.0 53.8 52.1 55.2 57.6 50.0 54.8 57.8 56.4 59.3 58.8 56.3 52.4 54.0 54.0 56.2 56.7 51.8 -5 8 2 63.4 57.1 58.3 56.3 58.3 53.1 54.6 58.2 51.0 58.0 61.1 70.4 56.2 58.2 52.2 64.2 59.8 67.7 50.7 60.0 60.7 68.0 62.3 6~.4 61.6 60.8 58.4 60.6 -._-
* Contaminated
Clin. Chim.
Acta,
with inorganic or organic iodine.
20 (1968) 113-126
GEL
THYROID
FUNCTION
INDIVtDUAL
VALUES
FILTRATION
IN
TEST
FOR
1x9
CHOLESTEROL,
WYPERTHYROID -“_.”
~__.._._ I
L 3 4 5 6 ii
UPTAKE,
[‘311]THYROIU
uptake __--.48 h gland
24 h gland
__..._--.185 IO.8
24 h urgne
48 h urine I.3 0.6
__ .._~_
72.4 90.5 77.3 59.6 82.5 31.0
IY”3 1x.4 x2.g 4.9 30.9 -1o.4
“_ 42.9 4.5.3 4z.4 48. x
2.0 3.9
35.5 35.9 34.3 31.6 30.2 38.8 34.2
39*4 36.6
r+a
‘84 IT8 ‘3-t
12.9 x3.8 14,s 22.3
Sr.3
77.1
j-8
400-3
40.3
27.3
12.1
35-9 38.0
77.1
73.6 87.2
IO.0 Il.3
0.7 0.5
45-o 33-7
27.7 42.6
72.2 49.7
4.2 I‘+“6 6.4 _.~”
4.2 7.8
38.4 35-7 43.1 34.8 35.3
30.4 45.1 39.fi 42.0
35.0 3r.4
42.3 47.7
187 193
24.0
89.9
I2 13 ‘4 15 16
226 1.36
12.0
78.9 71.3 -
9~4
27.6 .“._ 14.1
-
RESIN UPTAKE
28.8
--
10.2 1.4 0.5 ._. ---_. . with inorganic or organic iodine.
‘7 ‘54 IX* 219 ____..__~ * Contaminated
AND
GEL
FILTRATIOA
IN HYPERTHYROID
PROPYI.THLOlJRACIL
x0.
Beforc
2.8
-..
77*” _
IS.2
PATIENTS
BEFORE
treatment Protein-bouvzd
%
T, “,‘o
.
A fteer treatment L_--.___.. Resin @take 0’ ,I”
T, “/b
_--
20.0
60.5 68.5
3 4
24.2 22.7
55.9 -
5
XL%
39.4
20.2
62.9
2
4sj.O
40.2
AFTER
TREATMEX
T
Protein-bounh
30-4 3c.4 42-4 42.3
I
AND
30.7
-l_._ll_________
-P??B uptake I~.
I.2
3.0 o.h
38.8 38.4 31.6 35.0
21.5
-
IV
INDIVII)UAL VAl_‘UBS IN
FfLTRATiON
FOR
NYPOTWYROID
CHOLESTEROL,
FBI,
PATIENTS
[=l~]THYROID
Ckd-
PBI
[1311] Thyroid
ester01
@“&%
-__
mg % ----^
24 k gland
I 2 3 4 5 6
542 467 447 500 258 423
I.3 x.9 %“7 1.t I.0 a.8
7.8 11.7 6.3
206 325
L.3 “.5
10.6
rag
s9.5
-_
1.1
14.7
IS.+
322
-_
49.0 60.7
-
@take ‘7; --1--_-_ 48 J? 24 k wine gland “_. 6.9
I.0
S-5
___~
UPTAKE,
__.-I___
ivo.
B
.-
156
10 II
GEL
“/s
16.5 ix.4
20.0
I0
AND
115 I28
152
s7
UPTAKE
79.6 87.3 82.5 44.9 62.4 84.8 42.1
9
TABLE
RESIN
.~~-^-.“l.l..
[1311]Thyroid
No.
WITH
FBI,
PATIENTS
6O._y
II.8
59.4
5.2 -~ 0.42
55.4
5-5
I-._ 67.0
_._~
RESIN
UPTAKE
X?\‘D GEL
____l_---.
48k r, urzne
Resira uptake
__x____
Proteinbound
”/0
T, %
18.3 18.3 19.0 18.6 17.8 20.6
67.8 66.1 66.0 68.5 67.0 60.4
r3.9
19.2 18.g
67.5 67.g
$5.6 20.4
x9.4 x9.1
66.9 -._ . . _~
14.6 12.9 21.0 x9.6 -
Clin. Claim. Acfa,
20 (rg68)
rr3--126
JACOBS,
ZONIIAG
In 16 normal pregnant women the T, resin uptake values ranged from 15.1 to 17.674 (mean 16.4, SD. 0.8). In a patient whose pregnancy terminated in still-birth, a value of (16.5
20.8
was found. In 4 women receiving as can be seen in Table V.
the “pill”, the uptakeswere
low also
to 19,57/o),
TABLE
\i
ANI) GEL FILTRATION IN EUTHYROID AND W,X’ERTHYROID WOMEN DrRlSC ADMINISTRATION OF ORAI. CONTRACEPTIVES (LYNDIOL 2.5)
RESIN
UPTAKE
X0.
Cow.ditinn
lie.% “’ 0
I
euthyroid euthyroid euthyroid euthyroid
19. I 18.6 16.5
67.7
19.5
07.0
hyperthyroid hyperthyroid hyperthyroid
25.2 24.0
2 3 4 5 0
7
uptnire
Prcfsisz-bowzd 2’ 0 3 ,I> 08.0 _
00.9
27.j
serum @rote&s In patients with marked abnormalities in serum protein pattern the T, resin uptake test can give false results, with values suggesting hyperthyroidism in cases with normal thyroid function. It seems obvious that decreased levels of qlobulins, pre-albumin and albumin can cause increased resin uptake. From our material
Ab~wmal
(Table VI), this is indeed likely, although some exceptions (9, 16 of Table VI) occur. The increased uptake value of patient r3a was probably due to the very low serum albumin value: with increasing albumin concentration, the resin uptake decreased (13b). IIowever,
the limited data presented
sions concerning
this problem.
TAULE
here do not allow of any definite conclu-
VI
RESIP; UPTAKE VALUES IN EUTHYROID PATIEXTS WITH ABNORMAL SERUM PROTEIN
II.60
8.60 9.45 5.70 10.00 7.45 6.00 7.40 6.10 7.90 IO.50 5.80 4.80 4.30 3.90 3.60 4.90
c+ A- az-
g 0’ ;o
Globrrlil?~ g y* __-.. . .___
1 5s .?.IS 3.10
0‘45
0.30 0.15
3.45 3.85 3.35 3.40 3.60 3.05 2.90
0.q
3.85 3.85 0.50
0.25
x.25 0.75 0.6j 0.80 0.80
0.95 0.85 I.60 I.00
I.55 x.75 2.05
0.80
“-45
r.os
PATTERN
Diagnosis
Albumin
i.ZO
-.
Cirrhosis of the liver IgA-Kahkr IgA-Kahler IgA-Kahler IgG-Kahler IgG-Kahler IgA-paraproteinemia IgG-paraproteinemia lgG-paraproteinemia IgG-paraproteinemia Kahler Plasmocytoma Nephrotic syndrome Nephrotic syndrome Nephrotic syndrome Gastroenteropathy Generalized carcinomr
3 3 ..5 JO.5 40.9 31.1 28.7 2x.2 Xj.6 28.5
32.3 20.6 23.3 33.7 21.3 28.4 30.0 32.3
THYROID
FUNCTION
121
TEST
Sephadex gel filtration Most serum samples tested with the T, resin uptake test were investigated the Sephadex gel filtration technique as well (Tables I-VI). Most determinations carried out in duplicate. The SD., as calculated from duplicate determinations, 1.5%. Differences of up to 5% occurred, but were rare.
with were was
Normal values with this technique were in the range of 50 to 650/b. Of 48 euthyroid patients (Table I) 45 had values within this range, two receiving oral contraceptives were accordingly high (67.7?; and 68.0%) and theremaining patient (33 of Table I) had an unexplained high value of 70.4:/~. In hyperthyroidism (Table II) values of 27 to 48% were found, less than 5094 indicative of hyperfunction. Of the IO hypothyroid patients (Table IV), o had values between 66.0 and 68.5%. Patient 6, the one with a borderline resin uptake, had a gel filtration value of 60.4O,:,. In general, values above 659/o can be considered indicative of hypothyroidism. However, in pregnancy, high results are found as well (70 to 76:/,, mean 73.394,
S.D. 1.8:‘,)
Exfierimental The amount mental conditions,
and the same holds true for women taking the “pill” (Table V).
;1311]T, absorbed by the resin is dependent on several experiof L including temperature and time of incubation, homogenization
during
incubation, pH and buffer molarity of the mixture, amount of resin and with amount of inactive T3 (or T4) addede. In our experience and in accordance other+7, the uptake of T, by the resin increases with increasing temperature. If
incubation is carried out at room temperature in combination with shaking, uptake values are much lower and the difference between the highest and lowest values is only about half of that obtained at 37O. Incubation at 37O but without shaking, results in lower values as well (normal range about 15 to 20%) ; besides, the reproducibility appears to be rather poor in comparison with the proposed method. Required reproducibility
and optimal separation into the three clinically important classes was shaking during incubation. achieved at a temperature of 37O, under continuous Incubation during I h instead of 30 min or increasing the amount of resin per test, raises the resin uptake without improvement of the separation. The same holds true for addition of inactive T,. Most methods for the T, resin uptake include cumbersome washing of the resin. Repeated washings with isotonic saline (each time followed by centrifugation), up to 5 times in succession, are recommended. Apart from being time-consuming, washing involves the risk of loss of activity from the resin or loss of the resin itself. Therefore, attempts were made to avoid washing. From experiments made for this purpose it appeared that the remaining activity of the resin is less after each of the first 3 or 4 washings, only reaching a more or less constant level after some 4 to 6 washings. However, this was somewhat precarious. In a further study, including samples of normo-, hyper- and hypothyroid subjects, a number of estimations were carried out first without washing and then after three washings. In this way it could be shown that washing is not necessary and has no advantage. After three washings, the measured activities were found z to 30/o lower over the whole range of low, normal and high values, so that washing had no influence on the measure of separation. Clin. Chim.
Acta,
20 (1~68)
113-126
122
JACOBS, ZONDAG
The Sephadex gel filtration is based on the fact that protein-bound jrslI]TZ will pass relatively rapidly through the gel bed, unlike the free /131IjT,, which can only be &ted from the column with the aid of fluids having a strong affinity for T,, such as serum or plasma. The advantage of this column chromatographic technique over the resin uptake methods lies in the possibility of fractionation by means of separate collection of the effluent. Thus, in the case of contamination with radioactive iodide (as in samples from patients with previous administration of Nal”lI, or as in case the jlBfI]Ta preparation contains free radioactive iodide), it becomes possible to collect the [13r1]Ts from the column without elution of the more firmly bound iodide. However, in all our experiments, there appeared only one peak of activity in the effluent when eluting the column with buffer, representing the activity of the .1311]TZ bound by protein. So far as our !/ 1311]Ts preparation was contaminaied with iodide, this did not give a separate peak of activity. If, after elution with buffer, the remaining activity of the column was eluted with plasma, the recovery of the total activity applied to the column was in the range of 95.0 to 97.37;. The gel filtration technique was carried out roughly
according
to Cuar6n8.
In
preliminary experiments with fractionated collection of the effluent in I-ml portions, it appeared that the peak of activity is mainly obtained in the portions 3, L+,5 and 6. The first 2 ml running from the column contain no activity and can therefore be discarded. Routinely, the next 9 ml were collected and measured, representing the amount of [l”iI]T, bound by the serum proteins. The activity in the rest of the effluent was very low and can be neglected. Washing with 20 ml plasma was shown to be completely effective in removing the free /1311!T, from the column. Coarse-grained Sephadex was chosen, which has the advantage over the medium-grade quality of greater effluent flow (about z ml/min). The gel filtration process is rather temperature-sensitive.
With increasing
gel. In order to avoid inconstant a constant column temperature water jacket.
Pre-incubation
out at room temperature Some experiments
temperature
more T, is bound by the
results caused by fluctuations in room temperature of 37” was realized, using columns provided with a
of the serum samples with T, can equally well be carried
as at 37’; this does not make any difference. were carried out with the method described
by
Hvid
Hansen”“. This method can be considered as a resin uptake method, except that the resin is substituted by Sephadex. However, in our hands this technique was unsuccessful. After each washing, the remaining activity of the Sephadex was found less, even after 0 or 7 washings, without reaching a constant level. Reproducibilitywas less satisfactory and the uptake range was not sufficient for accurate measurements (below IO:,
of added activity).
COMPARISON
WITH OTHER METHODS AND DISCUSSION
Many thyroid function tests are now available for clinical practice. Since each test has its own advantages and pitfalls 13, it is not advisable to rely on a single procedure in thyroid testing. On the contrary, it can be stated that several different tests are needed in order to attain sufficient diagnostic precision. The [*311]thvroid uptake test is unique in so far as it gives a direct measure of thyroid activity ii U&IO.unfortunately, a number of extra-thyroid factors affect the results and there is a rather large overlap in physiological and pathological values,
which often makes their interpretation more or less uncertain. For example, for the 24-h thyroid uptake, a wide range of 30 to 70% was somewhat arbitrarily accepted as normal in our laboratory. Nevertheless, a 24-h uptake of over 70% can be found in euthyroid subjects with iodide deficiency (probably the case in patients 2,8,16,22, 25 and 36 in Table I), whereas values below 30% are encountered in normal patients patients I, 15 and 38 in Table I, and with iodide excess (iodide “contamination”; patient 18 in Table II). Normal values may be established at any time between 2 and 48 hours after administration of the tracer dose, and measurement at two or more different times gives more information, especially in view of the rapidity of 131f turnover. Routinely, the 24-and 48-h uptakes were measured in our laboratory, but in some instances a 6-h value is required as well, to establish the diagnosis of hyperthyroid&m. If an accelerated uptake is accompanied by rapid conversion of the lslI into the hormone form by the thyroid gland, then the maximum uptake value can be attained well within 24 h after 13iI administration. This can be illustrated by patient 4 of Table II : 6, 12,24,48 and 72-h thyroid uptakes, respectively, were 80.5%, 59.3%, 44.9%, 28.8% and 23.1%. The normal 24-h uptake values in the hyperthyroid patients 5,7 and g of Table II could be explained in the same way. The widely used determination of PI31 is generally considered to represent one of the most reliable indices of the state of thyroid function. We therefore compared the resin uptake test with the corresponding PI31 determinations. The results are shown in Fig. r. As can be seen from this figure, the correlation is good. A number of patients need further comment. Two subjects (8 and 30, Table I) had resin uptake values in the upper normal range, whereas the PBI of both serum samples was elevated, Clinically, it was doubtful whether they were euthyroid or hyperthyroid. Tn a third patient (33 of Table I) the elevated PBI was probably due to iodine contamination, as this patient was undoubtedly euthyroid, in accordance with the resin . ..m.2%6
Fig.
I.
Relationship between resin uptake and PBI, x = Contaminated with iodine.
r24
7(
JACOBS,
5.
A* ,
ZONDAG
‘*
.
5( 3a’ c z 0‘ J( )11 .t i 2 El a l(
Fig. 2. iodine.
Relationship
between gel filtration (protein bound TJ and PRI.
x = ~~~t~rn~nated with
uptake value (the gel ~ltration value of this patient, in the hypotl~yroid range, will be discussed later). The borderline resin uptake value of 20.6(~;~in a clinically distinctly hypoth~oid patient (No. 6 of Table IV) was somewhat too high in comparison with the very low PBT. Finally, in two patients clinically classified as normal (22 and 45 of Table I), a normal PBI was accompanied by low borderline resin uptakes of 20.5% and 20.6%. At least in one of them (No. 45) the assumption of a slight hypofunction seems justified, considering the rather high cholesterol level and the low [1*ll]thyroid uptake ipzvitro. As can further be seen from Fig. I, the resin uptake test permits the correct classification of patients in spite of iodine contamination. In Fig. 2, showing the ~ornpa~so~ between gel filtration results and PBX, nearly the same picture is seen, Since the correlation between resin uptake and gel filtration (Fig. 3) is very good, the resemblance between Figs. I and z is not surprising and requires no further ~o~l~lent. There is only one exception to be made. Patient 33, Table I, clinically euthyroid, presented a completely incomprehensil)le set of figures: high PBI (probably due to iodine contamination), normal resin uptake and hypothyroid gel filtration value. No explanation was found for this. Fig. 4 shows a summary of the resin uptake results in connection with the clinical classification of the patients. The separation between euthyroid and hyperthyroid patients is excellent; between euthyroid and hypothyroid patients the separation is probably slightly less sharp. Resin uptake values are always very low during normal pregnancy. As such, the test can be used as a kind of pregnancy test and can even play a part in predicting an abortion. In one patient, intrauterine foetal death had probably occurred. Three weeks later the resin uptake was zo.S%, supporting the diagnosis. The pregnancy terminated in St&birth. During the use of oral contraceptives, the values for resin uptake are also low. Clin. C?&. A&, 20 (18&H)I I.$-126
THYROID
FUNCTION
f;ig. 3. Relationship with iodine.
TEST
between gel filtration [protein bound T.J and resin uptake. x = C~nt~~ina~ed
If, on the other hand, resin uptake is normal in a patient taking the pill, this suggests ~lyperthyroidism~ as could be established in 3 patients (see also Table V). Finafly, it must be borne in mind that, in patients with grossly abnormal serum protein pattern, resin uptake values in the hyperthyroid range can be found which are not due to abnormal thyroid function, as could be demonstrated in IO out of 16 patients with dysproteinacmia. Fig. 5, summarizing the gel filtration resuIts
l
hype&h.
euth.
Fig. 4. Scatter diagram terine foetal death.
hypoth.
F-w
pill euth.
II hyperth.
disprot.
of resin uptake resutts in various clinical conditi~lls. Open circle I intrau-
.o8:.
d:
. --
:it :*
.A:.. .:jff:
l ..
.
0
~
I-
: . .. .*. .O-
1.
l
l
0
!
*
‘
hj/pert h.
cut h.
hypoth.
p=gn.
I PiII
euth.
Fig. 5. Scatter diagram of gel filtration results in various clinical conditions. terine foetal death.
Open circle = intrau-
in connection with the clinical classification, shows the same picture as Fig. 4 and leads to the same comments. In conclusion, the resin uptake test in the proposed modification has proved to be accurate, and particularly useful for the rapid screening of thyroid dysfunction. Although it is slightly more time-consuming, the same holds true for the Sephadex gel filtration technique. The advantage of both tests over PBI and [1311]thyroid uptake is vitro is that previous ingestion of iodine has no influence on the results. Furthermore, the resin uptake test does not require the administration of radioactive iodine to the patient. Tests can therefore be repeated at short intervals, and thus be of special value in following the effect of treatment. ACKNOWLEDGEMENTS We wish to express our gratitude to the Staffs of the Departments of Internal Diseases of the St. Elisabeth’s Hospital and the St. Joannes de Deo Hospital, Haarlem, for their cooperation in providing the clinical data of the patients. We also thank Dr. L. van Beugen for reading the manuscript and his helpful criticism and Miss A. Kruyt for her technical assistence. REFERENCES
1 J. ROBBINS AND 1. E. RX=, PhysioE. Rev., 40 (1960) 415. 2 S. H. INGBAR, J. C&n. Invest., 4~ (x963) 143. 3 M. W. HAMOLSKY, M. STEIN AND A. S. FXEEDBERG, I. CZin. Endocrinol. Metab., 17 (1957) 3.3. 4 M. L. MITCHELL, A. B. HARDEN AND M. E. O’ROURKE, 1. Clin. Endocrirzol. Mefab., 20 (1960) 5 G 7 8 ‘9 10
1474. Ii. STERLING AND M. TABACHNICK, J. Clin. Endocrinol. Metab., 21 (1961) 456. VI’. BBRNER, M. HOHBACH, E. MOLL AND H. BAYER, Klin. Wochschr., 44 (1966) 973. M. G. WOLDRING, A. BAKKER AND H. DOORENBOS, Acta Endocrinol., 37 (rg6r) 607. A. CV~R~N AND M. E. FUCUGAUCHI, Acta Endocrinol., 46 (1964) 161. I?. C.. SCRIBA, R. LANDGRAF, H. G. HEINZE AND K. SCHWARZ, Klin. Wochschr., 44 (1966) 69. I’. C. SCRIRA, H. G. HEINZE, R. LAXDGRAF, K. W. FREY AND K. SCHWARZ, Klin. Wochschr.,
xx
44 (1966) 131. R. GRKNICHER, A. BURGER, E. GFELLER AND H. STUDER, 2. Klin.
(1967) 1x7. IZ H. HSID HANSEN, Scaad. .J_ C&in. Lab. 13 J. C. Srsso~, J. Nzsclear Med., 6 (1965) Clin.
Chim.
Acta,
20 (1968)
rr3-126
Invest., 853.
18 (x966)
240.
Chem.
Klin.
&o&em.,
5