THE
THYROID
IN CHILDREN
II. In
Vitro
Erythrocyte
Uptake
of
Radioactive
L-Triiodothyronine
Ralph H. Kunstadter, M.D., Harvey Buchman, M.D., Morad Jacobson, M.D.,
and Leo Oliner, M.D.
Division of Pediatrics, Michael Reese Hospital and Medical Center, Chicago, the Radioisotope Service, Veterans Administration Hospital, and the Department of Medicine, Indiana University School of
Medicine, Indianapolis
ADDRESS: (R.H.K.) 664 N. Michigan Avenue, Chicago 11, Illinois.
27
PEDIATRICS, July 1962
T
HE IN vu-no UPTAKE of radioiodinatedtriiodothyronine by erythrocytes from whole blood has been reported as a useful index of thyroid function in the normal and in disease states.6 After 2-hour incubation,
erythrocyte uptake of triiodothyronine has been found to be increased in hyperthyroid patients while decreased levels were noted in hypothyroid ‘ Since the
var-ious in vitro erythrocyte uptake levels ob-tamed have been shown to be a function of the origin of the patient’s plasma and not that of the erythrocytes,7#{176} the quantitative availability and the degree of saturation of binding sites of plasma protein carriers for thyroid hormones have been proposed as the major determinants of the availability of thyroxin and/or triiodothyronine for up-take by erythrocytes or peripheral tis-sues.10-11
Although initial studies on eleetrophoret-ic separations of plasma protein in veronal buffer after addition of radiothyroxin indi-cated the binding protein to be located be-tween the alpha1-globulin and alpha2-glob-ulin (thyroxin-binding globulin, TBG) with albumin as a secondary carrier,1215 subse-quent studies in other buffer systems16’17 have provided data for a more complex sys-tem of protein-thyroid hormone interac-tion, so that at present there appear to exist two major protein carriers, namely, TBG
and a moiety with migration more anodal
than albumin (thyroxin-binding pre-albu-min, TBPA).16’17 While TBG binds both
thyroxin and triiodothyronine, the latter
more loosely, no association of triiodothy-ronine with TBPA has been shown,16 thus
the looser binding of triiodothyronine with
TBG
and
lack
of
it by TBPA accentuateany alteration in carrier protein availability and/or saturation when compared to
thy-roxin. Any thyroxin or triiodothyronine not
bound to the protein carriers would then
be present loosely bound to albumin or un-bound in the plasma for association with
erythrocytes or utilization by peripheral tissues.
In spite of the various factors affecting the procedure2b0.1S19 the erythrocyte
up-take of triiodothyronine can be utilized as a simple and rapid index of thyroid
func-tion and as such give information regarding
the quantitative availability of thyroid
hor-mone to the peripheral tissues. With this in
mind, the erythrocyte uptake of triiodothy-ronine was performed in a group of
chil-dren up to the age of 13 and a small group of normal and hyperthyroid adults. The
data to be presented indicate that the
up-take of triiodothyronine is elevated in chil-dren. The data also support our earlier
find-ings
from
J131kinetic
studies2#{176}on
radio-thyroxin utilization
and
turnover
studies21that a relative physiologic thyroidal
hy-peractivity exists in children when com-pared to adults.
Material
MATERIAL
AND
METHOD
Seventy normal children ranging from
newborn infants to 13 years of age were studied. All were well and free from acute
or chronic disease. Twelve normal and five
corn-TABLE I
ERYTHROCYTE UPTAKE OF THIIODOTIIYUONINE IN VARIOUS AGE GROUPS IN CHILDREN AND IN
EUTHYROID AND HYPERTHYROID ADULTS
28
parison. A child was considered normal if
his growth and development were normal
and there was no history of acute infection for 2 weeks prior to the study. Children with thyroid disorders were excluded, as were those who had received any thyroid interfering substance.
Method
The method is essentially that originally described by Hamolosky et al.’ 2 Venous
blood
samples
were drawn into a heparin-ized syringe or placed into a test tube con-taining a measured volume of heparin or powdered oxalate. (No apparent differences in erythrocyte uptake of triiodothyronine have been noted with use of heparin or oxalate.’) Radioiodine 1131 labeled1-tn-iodothyronine received from the supplier (Abbott Laboratories, Oak Ridge,
Tennes-see) as chromatographically pure was
di-luted with isotonic saline solution to con-tam 10-120 X 1O- ig/O.1 ml, with 100-400 counts/rninute/0.1 ml, and stored in the refrigerator and used for a period of 10 days. One-tenth milliliter of the diluted triiodothyronine was added to duplicate 2-ml whole blood samples in 10-ml enlen-meyer flasks. The flasks were stoppered and shaken at 37#{176}Cfor 2 hours. One-milliliter aliquots of the above were taken and the
saline-washed erythrocytes (5 times, with 10 ml at room temperature) of that aliquot were counted in a scintillation well-counter
for sufficient time to reduce counting error
below 2%. The erythrocyte uptake, in per cent, was calculated and corrected for a
hematocnit value of 100. The mean
differ-ence between duplicate determinations was 1.7 percentage points.
RESULTS
The results of the l-tniiodothyronine up-take levels at various ages in children and in adults are shown in Table I and Figure 1. The range and mean of the levels in the newborn period are greatly elevated as compared to those of euthyroid adults, with progressive decreases up to the age of 2 years; hereafter the mean levels are
essen-Group Age
Range
.Vum-ber
Th3 Uptake (%)
Range Mean (±S.D.) Children I 2 3 4 5 6 Newborn-3 days 1-Imo 1-4mo 27mo-44yr 5-9yr 104-lSyr 8 ii 8 19 15 9 .8-33.3 1I.-23.7 14.6-32.9 8.91.4 11.8-21.7 13.8-’22.2
28.l (± 3.9)
17.8(±3.9) ‘2.5(±6.3) 17.6(±5.5) 15.8(±’2.3) 17.’2(±2.8) Adults Euthyroid Hyperthyroid 12 s 9.0-16.5 19.O-i9.() 13.O(±2.6) 23.3 (±3.7)
tially the same but still above euthyroid adult levels. The difference in mean levels of all groups of children up to age 13 when compared to euthyroid adults are statisti-cally significant (p values < 0.01) as shown in Table II. The levels of “normal” new-born infants are equivalent to or higher than those of hyperthyroid adults. Corn-parison of the mean levels of the various children groups are shown in Table III. Al-though the difference in mean levels of groups up to age 5 years are statistically significant, the physiological significance of the lowered values from 1 to 12 months
is not readily apparent.
COMMENT
The data presented on erythrocyte up-take of 1-triiodothyronine in children up to age 13 years indicate a quantitatively greater availability of labeled
Range I- -I
Mean S. D. L\vR’t\w1
(19)
“I
F.’
04
C’)
.a
F-C)
(8) (8)
20
(15) (9)
10
(5)
0
F.’
1.1 (12)
0
11
F--
045.
AGE
-1-2
1-12 2-4.5 5-9 10-13
Newborn
MONTHS YEARS
Adults
Fic. 1. Erythrocyte radio-triiodothyronine uptake in children.
TABLE II
29
DIFFERENCES IN ERYTHROCYTE TRIIODOTHYRONINE UPTAKE VALUES IN THE
VARIOUS CHILDREN GROUPS WHEN COMPARED WITH EUTHYROID ADULTS
Group Age Range
Children Adults
U
Mean S.D. Mean S.D.
1
3
4
5 6
Newborn-S days
1-1mo
I4-4mo
7mo-44yr
5-9 yr
104-13 yr
8.05
17.83
.45
17.56
15.76 17.
3.86
3.88
6.31
5.52
.33 ‘2.83
13.04
13.04
13.04
13.04
13.04 13.04
.57
.57
.57
2.57
2.57
‘2.57
0 <0.001
0 <0.01
5 <0.001
50.5 <0.01
36 <0.01
14.5 <0.01
* For more detailed discussion of the U test, see Reference’2’2.
t One-tailed.
binding capacities in sera of children, par-ticularly in the newborn infant, where pro-tein concentration and content in the plasma are low relative to older children or adults, and/or looser association of thyroid hor-mones with TBG or TBPA.
Binding capacity of TBG for thyroxin in children aged 3 to 9 years has been re-ported to be essentially similar to that found in adults,23 while TBPA capacity was
15
0
found to be lower in males aged 13 to 16 when compared to adult men.24 If one can assume that TBPA binding capacity in chil-dren below age 13 is similar to that of the older age group, then the elevated 1-tn-lodothyronine erythrocyte uptake in the
group of children reported in our study
may reflect this alteration. (Meister et al.29
UPTAKE OF L-TRIIODOTHYRONINE
TABLE III
STATISTICAL SIGNIFICANCE OF DIFFERENCES IN
ERYTHROcYTE TRII0DOTHYR0NINE UPTAKE
VALUES IN VARIOUS CHILDREN GROUPS
Groups Compared
1&’2 ‘2&3
3&4
4&5
5&6
1 ‘23
43 1’26.5
45
<0.001 =0.05 <0.05 N.S. (>05)
N.S.
age 13 years as 59 ± 7
[S.D.J
tg/100 ml, as compared to 103 ± 3 p.g/100 ml in 14adults.) In euthyroid adults with various ill-nesses who had elevated erythrocyte tri-iodothyronine uptakes, the altered erythro-cyte uptake could be correlated with de-creased availability or capacity of binding sites of the TBPA carrier.11 Although this reduction in TBPA capacity may explain the increased thyroxin turnover in
chil-25 a more efficient system for
ex-tracting thyroid hormones at the cellular level in children cannot be ruled out. At present, the thyroid status in children then can be visualized as consisting of (a) in-creased thyroidal secretion2#{176} and (b) in-creased thyroxin 2526 coupled
with decreased TBPA binding capacity when compared to euthyroid adults. In contradistinction to the hyperthyroid state where these alterations are also noted, the relative thyroidal hyperactivity in children is a self-limiting one. Daily thyroxin de-gradation either in micrograms, or micro-gram/kilogram body weight, are similar to those for adult young men in children as early as 11 and 12 years of age.26
The differences in results obtained in this study, when compared to the two reports in children,27’28 bear comment. In these studies it is noted that data on erythrocyte uptakes in children,2 or in children after infancy,28 were similar to those reported in adults. Detailed data or ranking according to age are not presented in either report. No other apparent reason seems evident at this time
for the difference between our results on triiodothyronine erythrocyte uptake studies and these 728 since the methods used
were similar and the hematocnit values in our group were not elevated (range 28-42), to account for the higher values2S in our studies.
SUMMARY
The in vitro erythrocyte uptake studies of radioactive 1-tniiodothyronine in 70 nor-mal children, ranging in age from newborn infants to 13 years, are presented. The data reveal elevated uptake values in these chil-dren as compared to adults, indicating al-teration in binding capacity of thyroxin-binding serum protein carriers, in all prob-ability a reduction in thyroxin-binding pre-albumin capacity.
REFERENCES
1. Hamoisky, M. W., Stein, M., and Freedberg,
A. S. : The thyroid hormone-plasma protein
complex in man : II. A new in vitro method
for study of “uptake” of labeled hormonal components by human erythrocytes. J. Clin.
Endocr., 17:33, 1957.
2. Hamoisky, M. W., Golodetz, A., and
Freed-berg, A. S. : The plasma protein-thyroid
hormone complex in man: III. Further
studies on the use of the in vitro red blood
cell uptake of I’s’: l-triiodothyronine as a diagnostic test of thyroid function. J. Clin.
Endocr., 19:103, 1959.
.
Kurland, G. S., et al. : Thyroid function insupraventricular tachycardias; turnover of
intravenously infused I”-labeled thyroxine
and the red blood cell uptake of labeled 1-triiodothyronine. J. Clin. Endocr., 19:92, 1959.
4. Robbins, L. R. : Experience with the in vitro
erythrocyte uptake of I”-labeled
l-triiodo-thyronine in a routine clinical laboratory. J.
Clin. Endocr., 19:1292, 1959.
5. Ureles, A. L., and Murray, M.: The
erythro-cyte uptake of I’s’ labeled l-triiodothyronine as a measure of thyroid function. J. Lab.
Clin. Med., 54:178, 1959.
6. Friis, T.: In vitro uptake of J131 labeled
1-tn-iodothyronine by human enythrocytes. Acta.
Endocr., 33:117, 1960.
7. Hamolsky, M. W.: The plasma
protein-thy-roid hormone complex in thyrotoxicosis vs.
31
8. Fniis, T. : On the mechanism of the in vitro
uptake of I’’-labeled l-triiodothymnine by human erythrocytes. Acta Endocr., 33:134, 1960.
9. Christensen, K. L. : Trflodothyronine uptake by erythrocytes. Acta Med. Scand., 166:
141, 1960.
10. Wagner, H. N., Jr., Nelp, W. B., and Watts,
J. C. : Clinical and experimental evaluation
of the triiodothyronine (T1) red cell uptake test. Bull. Johns Hopkins Hosp., 108:161,
1961.
11. Ingbar, S. H., and Freinkel, N.: Regulation of
peripheral metabolism of the thyroid hor-mones. Recent Progr. Hormone Res., 16: 353, 1960.
12. Gordon, A. H., et al.: Nature of the circulating
thyroid hormone-plasma protein complex.
Nature, 169:19, 1952.
13. Deiss, W. P., Albright, E. C., and Larson, F.
C.: A study on the nature of the circulating
thyroid hormone in euthyroid and hyper-thyroid subjects by use of paper
electro-phoresis.
J.
Clin. Invest., 31:1000, 1952. 14. Robbins, J., and Rail,J.
E.: Zoneelectro-phoresis in filter paper of serum P’ after radioiodide administration. Proc. Soc. Exp.
Biol. Med., 81:530, 1952.
15. Robbins, J., and Rail, J. E.: The interaction of thyroid hormones and protein in biologi-cal fluids. Recent Progr. Hormone Res., 13:
161, 1957.
16. Ingbar, S. H.: Pre-albumin: a thyroxin-bind-ing protein of human plasma.
Endocrinol-ogy, 63:256, 1958.
17. Ingbar, S. H.: The interaction of the thyroid hormones with the proteins of human plas-ma. Ann. N.Y. Acad. Sci., 86:440, 1960.
18. Meade, R. C.: Possible errors in the
determi-nation of red blood cell uptake of
I’’-tniio-dothyronine.
J.
Clin. Endocr., 20:480, 1960.19. Tauxe, W. N., and Yamaguchi, M. Y. :
Van-ants affecting results of red cell
triiodothy-ronine uptake test. Amer. J. Clin. Path., 36:1, 1961.
20. Oliner, L., Kohlenbrenner, R. M., and
Kim-stadter, R. H. : Thyroidal secretion and deg-radation rates in children. J. Dis. Child.,
96:536, 1958.
21. Oliner, L., et al.: Radiothyroxin utilization in children. J. Lab. Clin. Med., 56:933, 1960.
22. Siegel, S. : Nonparametnic statistics for the
behavioral sciences. New York, McGraw-Hill, 1956.
23. Haddad, H. M.: Studies on thyroid hormone
metabolism in children.
J.
Pediat., 57:391,1960.
24. Dreyer, D. J., and Man, E.: Thyroxin-binding
protein studies in normal male adolescents.
Abstracts of American Goiter Association
Meeting, Philadelphia, May, 1961, p. 7.
25. Haddad, H. M.: Rates of I’ labeled thyroxine metabolism in euthyroid children.
J.
Clin. Invest., 39:1590, 1960.26. Oliner, L., et al.: The thyroid in children: IV.
Thyroxin degradation and turnover studies. In preparation.
27. Cnigler,
J.
F., Hertz, J., and Hamolsky, M. W.: In vitro red cell uptake of P31-l-triiodothyro-nine as a measurement of thyroid function in children.J.
Dis. Child., 98:665, 1959. 28. Marks, J., Wolfson, J., and Klein, R.: Neonatalthyroid function, erythrocyte T uptake in
early infancy. J. Pediat., 58:32, 1961. 29. Meister, L., et al.: Studies on thyroxin binding
