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Neonatal thyroid function: erythrocyte T3 uptake in early infancy
32
January 1961
T h e Journal o[ P E D I A T R I C S
Neonatal thyroid function: erytlorocyte uptake in early / /ancy j. Marks, M.D., J. Wolfson, M.D., and R. Klein, M.D. ~ PITTSBURGH~
PA.
S E v E 1~A L reports have contained evidence suggestive of increased thyroid function in the neonatal period. Elevated protein-bound iodine values were reported by Danowski? These were highest on the third day of life but fell precipitously during the first six weeks of life, and then more slowly until adult levels were reached at the end of the first year. M a n 2 found similar changes in the butanol extractable iodine values. In 1954, Van Middelsworth 3 demonstrated that the thyroid uptake of i 131 in 7 normal 2- to 3-day-old males was significantly above adult levels. Both PBI and 1lal uptake values, however, may be elevated in certain types of hypothyroidism and in euthyroid individuals with goiters. ~, 5 The introduction by Hamolsky ~ of the measurement of erythrocyte triiodothyronine uptake as an index of thyroid function afforded the opportunity of investigating another parameter of neonatal thyroid function. The present study
From the Department o[ Pediatrics, University of Pittsburgh School of Medicine, Children's Hospital of Pittsburgh, and Elizabeth Steel Magee Hospital. Study supported in part by the Health Research and Services Foundation, Grant A-II. ~'Address, Department o] Pediatrics, Children's Hospital oJ Pittsburgh, Pittsburgh 13, Pa.
was designed to evaluate the possibility of increased thyroid-binding globulin in the newborn as a cause for the increase in protein-bound iodine, and to establish that these changes were not solely the result of transplacentally transfused material.
METHODS Hamolsky's procedure for measuring the in vitro red cell uptake of II~l-labeled triiodothyronine was utilized. In this method 36 - 144 x 10-~ mcg. of triiodothyronine in 0.1 ml. volume is incubated at 37 ~ C. for 2 hours with 3 ml. of whole blood. The specimens are continuously agitated during this period. Following incubation, two 1 ml. aliquots are counted in an appropriate welltype scintillation counter. The red cells are then washed with 10 ml. volumes of isotonic saline five times, resuspended in 1 ml. saline, and counted. T h e hematocrit is determined simultaneously for each specimen. After necessary corrections fo'r background are made, the amount of radioactivity in the erythrocyte, corrected to 100 per cent hematocrit, is compared to that of the whole blood and multiplied by 100. This is labeled per cent triiodothyronine or T3 uptake. Heparinized blood was used throughout our studies. T h e mean difference between the
Volume 58 Number 1
Neonatal thyroid [unction
two 1 ml. aliquots counted was less than 5 per cent. In a preliminary experiment, the erythrocyte T:~ uptake was measured in blood obtained from normal adults after separation of the cells and plasma and reconstitution of the blood so as to produce aliquots with roughly 25, 50, and 75 per cent hematocrits. To these was added 72 x 10-4 mcg. of radioactive triiodothyronine. In one experiment measurements were also made with 36 and 144 x 10-* mcg. of T3. In another single experiment the red cells were washed with 5 and 15 ml. aliquots of saline as well as with 10 ml. volumes. Subsequently all assays were carried out by the Hamolsky technique, which was modified so that the hematocrit of the blood assayed was reconstituted to a range of 42 to 50 per cent. The following groups of subjects were utilized: Adult laboratory personnel, hospitalized children 1 ~ to 15 years of age, full-term and premature infants of various ages, and pregnant women at delivery. The older children were free from obvious endocrine abnormality and from any acute illness. The infants were born of normal deliveries, and were considered healthy upon examination in the lying-in hospital or at the well-baby clinics where the blood was obtained. Blood was obtained at delivery from 7 mothers and from the cords of their infants. Repeat determinations on the same infants were made at 3 to 5 days of age. In addition to the erythrocyte radioactive Ta uptake, butanol extractable iodine (BEI) ~ was also measured in some cases. RESULTS
A number of blood specimens from adults were adjusted to three different hematocrits and the uptakes compared for the three hematocrits for each specimen. As noted from Table I, the uptakes for the adjusted high hematocrits are higher and ~hose for the adjusted low hematocrits lower than those observed for specimens of the same
"~BEI determinations were ratories, Los Angeles~ Calif.
m a d e by Bio-Seience
Labo-
33
Table I. Effect of hcmatocrit on per cent erythrocyte Ta uptake. Blood specimens from 4 normal adults reconstituted to three arbitrary hematocrits each
Subjects I
II III IV
74-76 % 14.5~ 24.4 23.8 29.0
Hematocrit 46-49 % 11.4 18.3 14.0 17.9
22-26 % 8.2 11.1 10.3 13.3
"x'54 hematoerit.
blood at a normal hematocrit. Fourfold variation in the amount of radioactive T8 employed, and the threefold changes in the volume of the saline wash did not affect the test results. Table I I and Fig. 1 portray results from one experiment where aliquots of a sample of "normal" adult blood were reconstituted to hematocrits of 22, 48, and 76 per cent. The per cent erythrocyte Ta uptakes for the three levels of hematocrits were 10, 15, and 24 per cent. No significant difference in uptake was produced in the 48 per cent hematocrit specimen by varying the dose of T8 added from 36 to 72 to 144 x 10`4 mcg. Table I I I gives the results of a similar experiment in which one dose of radioactive T3 was used, but the volume of the saline washes was varied from 10 to 15 c.c. for the high hematocrit and to 5 ml. for the low hematocrit. As may be seen, this had no perceptible effect. A small group of normal adults and a larger group of hospitalized children, aged 1 89 to 15 years, were studied. The mean erythrocyte T3 uptake for 8 normal adults and 40 older children without known disease was 13.4 and 13.9 per cent, respectively. This level does not differ significantly from the normal euthyroid range for adult and juvenile subjects reported by Hamolsky s, 7 and by Crigler 8 of 10 to 17 per cent with a mean uptake of 13.9 per cent. Table IV records the per cent Ta uptake of healthy, full-term infants. The mean value for cord blood, 16.3 per cent uptake, was
34
Marks, WoI/son, and Klein
January 1961
significantly greater than that shown for the older children with a probability of less than 0.01 of this being due to chance. The mean of older, well, full-term infants 3 to 9 days of age was 19.8 per cent, which is again significantly greater than the cord blood value. At 2 to 4 weeks of age, the mean Ta uptake was significantly less than that at the latter part of the first week (p < .05), but it was still higher than that of normal, older children (p < .01). The mean level of T~ uptake for the group of patients 6 to 9 weeks of age was in the normal range for older children and adults and was significantly lower than that of the 2- to 4-week-old newborn infants. Thus, the per cent Ta uptake reaches a maximum in the first week of life and fails to within normal adult values by 6 to 9 weeks of age. Fig. 2 portrays the mean T3 uptake for fullterm, normal newborn infants at different periods of life as compared to the mean and range for normal older children and adults.
Table II. Effect of hematocrit and dose of triiodothyronine (T3) on per cent erythrocyte Ta uptake. Blood from a single adult reconstituted to three arbitrary hematocrits and incubated with variable Ta dosage.
Dose of T~ (3" x 10-5)
76%
144 72 36
26.8 24.5 23.7
j
Hematocrit 48% 1 22.5% 15.3 14.0 15.0
10.1 10.3 8.0
Table III. Effect of hematocrit and of volume of isotonic saline wash on per cent erythroeyte T3 uptake. Blood from a single adult adjusted to three different hematocrits and washed with variable amounts of normal saline
Volume saline wash 15 c.c. 10 c.c. 5 c.c.
74% 29.3 29.0 -
-
Hematocrit [ 47% I 26% -17.9 -
-
-13.3 13.0
EFFEGT OF HEMATOGRIT "
25.
ON R B C T 3 U P T A K E
T ~ AODED
=
9
: 3,,~,,o-',
":
~ - ~ , O "t'Z~ x I0 -'i = ~ 14415x I0 -'ll
/* " / ,r ,./"/~/
/ /,4 ////7 9 ;';// ..7" ,i/
15-
10-
,o
~'o
3'o
go
5o
~o
io
8'o
HEMATOCRIT
Fig. 1. Per cent erythrocyte T~ uptake of 9 aliquots of blood reconstituted from a single sample from a normal adult.
Table V and Fig. 3 present the results of similar measurements as well as measurements of butanol extractable iodine in a group of mothers and their infants. Blood was obtained from both the mother and the infant's cord at delivery and again from the infant at 3 to 5 days of age. Cord erythrocyte T3 uptake was significantly higher than the maternal T3 uptake, while that for the 3- to 5-day-old infants was still higher. Similar findings were also observed in another group of 11 paired cord and infant specimens. Erythrocyte T3 uptake for the mothers was 11.3 per cent, which is significantly lower than that of normal adults, corroborating Hamolsky's~ observations. Parallel determinations of BEI are recorded for 5 of the 7 sets of infants studied in this special group. It will be noted from the values shown in the table that the BEI's for the cord bloods are high but distinctly less than the maternal values, while there is again an increase at 3 to 5 days. Thus, the relationship of the BEI to per cent T3 uptake in maternal blood differs from that in infant cord blood. Premature infants were then compared with full-term infants. Eleven healthy prematures 3 to 9 days of age showed a mean uptake of 26.5 per cent, compared to a mean of 19.8 per cent for full-term infants of a
Volume 58
Number 1
N e o n a t a l thyroid [ u n c t i o n
similar age. Nine prematures, 2 to 4 weeks of age, had a m e a n of 25.0 per cent, as opposed to 17.5 per cent for the full-term infants of the same age. T h e m e a n for prem a t u r e infants 6 to 9 weeks of age, 23.3 per cent, differs from the mean of 13.2 per cent for full-term infants of the same age. T h e differences between all three pairs are significant; the probability of this being due to chance is less than 0.01. Thus, the level of uptake in premature infants is higher than the level of uptake in full-term infants at the same age (Table V I and Fig. 4). Butanol extractable iodine values in 5 premature newborn infants were not higher than those in full-term infants.
Table IV. Per cent erythrocyte Ts uptake of full-term infants Significance
N o . o'f
sub-
Mean
4"
standard
Comparison with preceding age group
Comparison with "'normal" children [rom 11/2 to 15 years
Age
jects
error
Cord 3 to9 days 2 to4 weeks 6 to9 weeks
19
16.3+-0.7
56
19.8+0.5
p < 0.01
p ~ 0.01
18
17.5 +-0.7
p ~ 0.05
p < 0,01
24
13.2 +0.8
p ~ 0.01
p ~ 0.1
--
p ~ 0.01
DISCUSSION Hamolsky 7 has offered the following explanation of the mechanism of the test. Although several plasma protein elements bind thyroid hormone, it is believed that one, an alpha globulin, binds material more firmly than the others. E r y t h r o c y t e Ts uptake is apparently directly related to the degree of saturation of alpha-globulin binding sites by thyroxin-like material. W h e t h e r the effect noted is a specific competitive relationship between the red cell and the plasma binding sites, or whether this p h e n o m e n o n is a result of saturation and overflow is as yet somewhat uncertain. I n any event, for practical
3 5
Fig. 2. S E M ~- Standard error of the mean. Dotted lines represent 2 standard errors above and below the mean for 40 "normal" children, 11/2 to 15 years of age.
purposes, it could be postulated that in hyperthyroidism m o r e of the presumed binding sites would be occupied so that the added triiodothyronine would be attached to the more loosely b o u n d sites, and thence to the surface of the red cells producing the elevated erythrocyte Ta uptake. T h e converse would occur with hypothyroidism. A l t h o u g h triiodothyronine uptake on the red cell usually parallels other parameters of thyroid function, there are situations where the protein-bound iodine is elevated and the triiodothyronine uptake is depressed. An example of this is found in pregnancy, where there is a p r i m a r y increase in thyroidbinding globulin and an elevated circulating PBI secondary to the increased binding capacity. I n g b a r 9 has shown that thyroid-binding protein is greatly increased during pregn a n c y and to a lesser but significant degree in cord plasma. Thus, red blood cell T~
Table V. Per cent erythrocyte Ta uptake in a paired series of mothers and their infants
T3 Uptake
Significance
Mother 11.3 + 0.4 -Cord 14.8+0.9 p ~ 0 . 0 1 Infant 21.3+0.9 p ~ 0 . 0 1
BEI (meg.~ 100 ml.)
Significance
8.1 + 0.4 -5.6+0.5 p ~ 0 . 0 1 9.1+0.7 p ~ 0 . 0 1
3 6 Marks, Wolfson, and Klein
January 1961
Table VI. Per cent erythrocyte T3 uptake of full-term and premature infants
age 3 to 9 days 2 to 4 weeks 6 to 9 weeks
I
FuZZ-ter,~infant, 19.8_+0.5 (56) e 17.5+0.7 (18) 13.2 +0.8 (24)
I
eremature infants 26.5+1.2 ( l l ) t 25 +1.6 (9) 23.3+- 1.9 (5)
l
Signi~can. p<0.01 p<0.01 p < 0.01
"~Number of subjects in parentheses. t T h e m e a n B E I for 5 of these p r e m a t u r e newborn infants is 6.5 meg. per 100 ml. with a S.E.M. of • 0.3.
uptake probably reflects two factors: (1) the level of circulating thyroid-binding protein, and (2) its relative level of saturation by circulating thyroid hormone. One could postulate that in a specimen with a low hematocrit the larger plasma volume would contain relatively more thyroidbinding protein and consequently erythrocyte T3 uptake values would be falsely low. Our results showing an increase in erythrocyte Ta uptake parallel to an increase in hematocrit afford some evidence that this m a y be so. Other explanations are possible, however. Among these was one suggested by the work of Meade, 1~ who reported that erythrocyte T3 uptake was inversely proportional to the amount of washing of the red cells. However, we were unable to find any evidence to confirm this possibility in the single experiment carried out with different volumes of saline washes. Because of the wide variation in hematocrit seen in early infancy, all assays in the body of this report were carried out by the modification of Hamolsky's technique with the hematocrit adjusted to within the adult range (from 42 to 50 per cent) at the start of the procedure. When Danowski 1 first reported on his findings in 1951, he discussed several different possible explanations of the elevated protein-bound iodine noted in the first year of life: (1) that this represented a true increase in thyroid function, and (2) that the findings could be explained by changes in extracelIular fluids usually seen in newborn infants. Danowski discarded the latter theory, however, because this would not explain the persistence of elevation throughout as long a period as he demonstrated. In addition, the magnitude of changes in fluid volume are
not great enough to explain the elevations of protein-bound iodine. Danowski also raised the possibility that the infant metabolized thyroid hormones at a slower rate. This seems unlikely, however, in view of the work of Stevenson, 11 who showed that premature infants could ingest 120 to 180 rag. thyroid daily without clinically manifest toxicity. Another explanation for the elevated PBI or BEI, not discussed by Danowski, is in terms of some hormonally inert iodinated protein. At the present time, however, investigation of this possibility is not feasible since it would require the in vivo use of radioisotopes in newborn infants. Danowski gives two other possibilities: (1) that the elevated PBI is secondary to an increased plasma thyroid-binding capacity, and (2) that it is a reflection of transplacentally transfused material. I t will be noted that there is a difference between the triiodothyronine uptake and the protein-bound iodine in the newborn as corn-
Fig. 3. Mean + standard error for per cent T~ uptake shown at bottom of graph.
Volume 58
Number 1
N e o n a t a l thyroid f u n c t i o n
% R B C T~. U P T A K E IN T E R M AND PREMATURE INFANTS
3 7
of normal for full-term newborn infants, suggests that the thyroid-binding protein is less elevated in the premature than in the fullterm infant.
:30-
25
SUMMARY 2o
- - 2 SEM
+2SEM
15
B,
, ~ -~:a PREMATURE FULL T E R M
--2
I0 AGE-
Fig. 4. S E M
i
=
5 - 9 DAYS
z
2-4
WEEKS
-MEAN
SEM i
6~9 WEEKS
Standard error of the mean.
pared with that of the mother. I n the maternal serum, the PBI or B E I is elevated secondarily to an increased thyroid-binding protein as was previously demonstrated by Ingbar. 9 Both our findings and those of Hamolsky, 6 however, show a depression of the per cent T3 erythrocyte u p t a k e in maternal blood. I n addition, observations show that the cord blood obtained from the infants of the same mothers has a somewhat smaller BEI but an erythrocyte Ta uptake which is significantly greater both than that of the mothers and that of normal older children or adults. These changes show a significant discrepancy between the normal and umbilical cord findings. Thus, it is strongly suggested that the changes observed are more than a reflection of maternal thyroid status in the infant. T h e finding of an elevated erythrocyte T3 uptake in the newborn makes it seem impossible to explain the concurrently elevated PBI or B E I solely on the basis of an elevated thyroid-binding protein. If the increased PBI were secondary to an increased thyroid-binding capacity, one would expect a low T3 uptake and a high PBI, as seen in pregnancy. O u r findings, coupled with those of Danowski and of V a n Middelsworth, strongly suggest that there is a true increase in thyroid function in the newborn period. T h e finding of a more elevated Ta uptake in the premature, who has a BEI within the range
1. T h e erythrocyte Ta uptake method of Hamolsky has been modified so that it is adaptable to individuals in the pediatric age range. 2. Using this technique, we have shown elevated erythrocyte Ta values in the fullterm neonate. These fall to within the adult range when the infant is 6 to 9 weeks of age. T h e elevation of erythrocyte Ta uptake appears more m a r k e d and more prolonged in the premature infant. 3. T h e elevated erythrocyte Ta uptake shows that the increased protein-bound iodine o r butanol extractable iodine of the neonate is not the result of an increased thyroid-binding protein. T h e different relationships of the Ta uptake to the BEI in the maternal and in the cord blood specimens indicate t h a t the observations in the newborn are not merely a reflection of maternal thyroid status. O u r d a t a suggest that there is a true increase in thyroid secretion in the newborn infant. REFERENCES 1. Danowski, T. S., Johnson, S. Y., Price, W. C., MeKelvey, M., Stevenson, S. S., and McCluskey, E. R.: Protein Bound Iodine in Infants From Birth to One Year of Age, Pediatrics 7: 240, 1951. 2. Man, E. B., Picketing, D. E., Walker, J., and Cooke, R. E.: Butanol Extractable Iodine in the Serum of Infants, Pediatrics 9: 32, 1952. 3. Van Middelsworth, L.: Radioactive Iodine Uptake of Normal Newborn Infants, A. M. A. Am. J. Dis. Child. 88: 439, 1954. 4. Werner, S. C., Block, R. J., Mandl, R. H., and Kassenaar, A. A. H.: Pathogenesis of a Case of Congenital Goitre With Abnormally High Levels of SPI and With Mo.no and Diiodotyrosine in the Serum, J. Clin. Endocrinol. 17: 817, I957. 5. Stanbury, J. B., and Hedge, A. N.: A Study of a Family of Goitrous Cretins, J. Clin. Endocrinol. 10: 1471, 1950. 6. Hamolsky, M. W., Stein, M., and Freedburg, A. S.: The Thyroid Hormone-Plasma Protein
38
Marks, Wol[son, and Klein
Complex in Man, II. A New in Vitro Method for Study of "Uptake" of Labeled Hormonal Components by Human Erythrocytes, J. Clin. Endocrinol. 17: 33, 1957. 7. Hamolsky, M. W., Godoletz, A., and Freedberg, A. S.: The Plasma Protein-Thyroid Hormone Complex in Man, III. Further Studies of the in Vitro Red Blood Cell Uptake of Ilal-l-Triiodothyronine as a Diagnostic Test of Thyroid Function, J. Clin. Endocrinol. 19: 103, 1959. 8. Crigler, J. F., Hertz, J., and Hamolsky, M. W.: In Vitro Red Blood Cell Uptake of I13M-Triiodothyronine as a Measuremen.t of
January 1961
Thyroid Function in Children, A. M. A. J. Dis. Child. 98: 665, 1959. 9. DoMing, J. T., Freinkel, N., and Ingbar, S. H.: Thyroxin-Binding by Sera of Pregnant Women, Newborn Infants and Women With Spontaneous Abortion, J. Clin. Invest. 35: 1263, 1956. 10. Meade, R. C.: Possible Errors in Determination of RBC Uptake of 1131 Triiodothyronine, J. Clin. Endocrinol. 20: 480, 1960. 11. Stevenson, S. S., Wirth, P., Bastiani, R., and Danowski, T. S.: Some Effects of Exogenous Thyroid or Thyroxine Upon Premature Infants, Pediatrics 12: 263, 1953.
A strange cause of death English newspapers have recently reported the death of a 9-year-old boy caused by inhaling gasoline fumes. For some time the parents had observed the strange attraction of the boy [or gasoline fumes and had reprimanded and punished him. On the fatal day, while his father was working nearby, the boy crept under a canvas sheet which covered his father's motorcycle and removed the cap from the gasoline tank. After being missed [or three hours he was discovered dead with his face beside the open gasoline tank.
January 1961
T h e Journal o[ P E D I A T R I C S
Neonatal thyroid function: erytlorocyte uptake in early / /ancy j. Marks, M.D., J. Wolfson, M.D., and R. Klein, M.D. ~ PITTSBURGH~
PA.
S E v E 1~A L reports have contained evidence suggestive of increased thyroid function in the neonatal period. Elevated protein-bound iodine values were reported by Danowski? These were highest on the third day of life but fell precipitously during the first six weeks of life, and then more slowly until adult levels were reached at the end of the first year. M a n 2 found similar changes in the butanol extractable iodine values. In 1954, Van Middelsworth 3 demonstrated that the thyroid uptake of i 131 in 7 normal 2- to 3-day-old males was significantly above adult levels. Both PBI and 1lal uptake values, however, may be elevated in certain types of hypothyroidism and in euthyroid individuals with goiters. ~, 5 The introduction by Hamolsky ~ of the measurement of erythrocyte triiodothyronine uptake as an index of thyroid function afforded the opportunity of investigating another parameter of neonatal thyroid function. The present study
From the Department o[ Pediatrics, University of Pittsburgh School of Medicine, Children's Hospital of Pittsburgh, and Elizabeth Steel Magee Hospital. Study supported in part by the Health Research and Services Foundation, Grant A-II. ~'Address, Department o] Pediatrics, Children's Hospital oJ Pittsburgh, Pittsburgh 13, Pa.
was designed to evaluate the possibility of increased thyroid-binding globulin in the newborn as a cause for the increase in protein-bound iodine, and to establish that these changes were not solely the result of transplacentally transfused material.
METHODS Hamolsky's procedure for measuring the in vitro red cell uptake of II~l-labeled triiodothyronine was utilized. In this method 36 - 144 x 10-~ mcg. of triiodothyronine in 0.1 ml. volume is incubated at 37 ~ C. for 2 hours with 3 ml. of whole blood. The specimens are continuously agitated during this period. Following incubation, two 1 ml. aliquots are counted in an appropriate welltype scintillation counter. The red cells are then washed with 10 ml. volumes of isotonic saline five times, resuspended in 1 ml. saline, and counted. T h e hematocrit is determined simultaneously for each specimen. After necessary corrections fo'r background are made, the amount of radioactivity in the erythrocyte, corrected to 100 per cent hematocrit, is compared to that of the whole blood and multiplied by 100. This is labeled per cent triiodothyronine or T3 uptake. Heparinized blood was used throughout our studies. T h e mean difference between the
Volume 58 Number 1
Neonatal thyroid [unction
two 1 ml. aliquots counted was less than 5 per cent. In a preliminary experiment, the erythrocyte T:~ uptake was measured in blood obtained from normal adults after separation of the cells and plasma and reconstitution of the blood so as to produce aliquots with roughly 25, 50, and 75 per cent hematocrits. To these was added 72 x 10-4 mcg. of radioactive triiodothyronine. In one experiment measurements were also made with 36 and 144 x 10-* mcg. of T3. In another single experiment the red cells were washed with 5 and 15 ml. aliquots of saline as well as with 10 ml. volumes. Subsequently all assays were carried out by the Hamolsky technique, which was modified so that the hematocrit of the blood assayed was reconstituted to a range of 42 to 50 per cent. The following groups of subjects were utilized: Adult laboratory personnel, hospitalized children 1 ~ to 15 years of age, full-term and premature infants of various ages, and pregnant women at delivery. The older children were free from obvious endocrine abnormality and from any acute illness. The infants were born of normal deliveries, and were considered healthy upon examination in the lying-in hospital or at the well-baby clinics where the blood was obtained. Blood was obtained at delivery from 7 mothers and from the cords of their infants. Repeat determinations on the same infants were made at 3 to 5 days of age. In addition to the erythrocyte radioactive Ta uptake, butanol extractable iodine (BEI) ~ was also measured in some cases. RESULTS
A number of blood specimens from adults were adjusted to three different hematocrits and the uptakes compared for the three hematocrits for each specimen. As noted from Table I, the uptakes for the adjusted high hematocrits are higher and ~hose for the adjusted low hematocrits lower than those observed for specimens of the same
"~BEI determinations were ratories, Los Angeles~ Calif.
m a d e by Bio-Seience
Labo-
33
Table I. Effect of hcmatocrit on per cent erythrocyte Ta uptake. Blood specimens from 4 normal adults reconstituted to three arbitrary hematocrits each
Subjects I
II III IV
74-76 % 14.5~ 24.4 23.8 29.0
Hematocrit 46-49 % 11.4 18.3 14.0 17.9
22-26 % 8.2 11.1 10.3 13.3
"x'54 hematoerit.
blood at a normal hematocrit. Fourfold variation in the amount of radioactive T8 employed, and the threefold changes in the volume of the saline wash did not affect the test results. Table I I and Fig. 1 portray results from one experiment where aliquots of a sample of "normal" adult blood were reconstituted to hematocrits of 22, 48, and 76 per cent. The per cent erythrocyte Ta uptakes for the three levels of hematocrits were 10, 15, and 24 per cent. No significant difference in uptake was produced in the 48 per cent hematocrit specimen by varying the dose of T8 added from 36 to 72 to 144 x 10`4 mcg. Table I I I gives the results of a similar experiment in which one dose of radioactive T3 was used, but the volume of the saline washes was varied from 10 to 15 c.c. for the high hematocrit and to 5 ml. for the low hematocrit. As may be seen, this had no perceptible effect. A small group of normal adults and a larger group of hospitalized children, aged 1 89 to 15 years, were studied. The mean erythrocyte T3 uptake for 8 normal adults and 40 older children without known disease was 13.4 and 13.9 per cent, respectively. This level does not differ significantly from the normal euthyroid range for adult and juvenile subjects reported by Hamolsky s, 7 and by Crigler 8 of 10 to 17 per cent with a mean uptake of 13.9 per cent. Table IV records the per cent Ta uptake of healthy, full-term infants. The mean value for cord blood, 16.3 per cent uptake, was
34
Marks, WoI/son, and Klein
January 1961
significantly greater than that shown for the older children with a probability of less than 0.01 of this being due to chance. The mean of older, well, full-term infants 3 to 9 days of age was 19.8 per cent, which is again significantly greater than the cord blood value. At 2 to 4 weeks of age, the mean Ta uptake was significantly less than that at the latter part of the first week (p < .05), but it was still higher than that of normal, older children (p < .01). The mean level of T~ uptake for the group of patients 6 to 9 weeks of age was in the normal range for older children and adults and was significantly lower than that of the 2- to 4-week-old newborn infants. Thus, the per cent Ta uptake reaches a maximum in the first week of life and fails to within normal adult values by 6 to 9 weeks of age. Fig. 2 portrays the mean T3 uptake for fullterm, normal newborn infants at different periods of life as compared to the mean and range for normal older children and adults.
Table II. Effect of hematocrit and dose of triiodothyronine (T3) on per cent erythrocyte Ta uptake. Blood from a single adult reconstituted to three arbitrary hematocrits and incubated with variable Ta dosage.
Dose of T~ (3" x 10-5)
76%
144 72 36
26.8 24.5 23.7
j
Hematocrit 48% 1 22.5% 15.3 14.0 15.0
10.1 10.3 8.0
Table III. Effect of hematocrit and of volume of isotonic saline wash on per cent erythroeyte T3 uptake. Blood from a single adult adjusted to three different hematocrits and washed with variable amounts of normal saline
Volume saline wash 15 c.c. 10 c.c. 5 c.c.
74% 29.3 29.0 -
-
Hematocrit [ 47% I 26% -17.9 -
-
-13.3 13.0
EFFEGT OF HEMATOGRIT "
25.
ON R B C T 3 U P T A K E
T ~ AODED
=
9
: 3,,~,,o-',
":
~ - ~ , O "t'Z~ x I0 -'i = ~ 14415x I0 -'ll
/* " / ,r ,./"/~/
/ /,4 ////7 9 ;';// ..7" ,i/
15-
10-
,o
~'o
3'o
go
5o
~o
io
8'o
HEMATOCRIT
Fig. 1. Per cent erythrocyte T~ uptake of 9 aliquots of blood reconstituted from a single sample from a normal adult.
Table V and Fig. 3 present the results of similar measurements as well as measurements of butanol extractable iodine in a group of mothers and their infants. Blood was obtained from both the mother and the infant's cord at delivery and again from the infant at 3 to 5 days of age. Cord erythrocyte T3 uptake was significantly higher than the maternal T3 uptake, while that for the 3- to 5-day-old infants was still higher. Similar findings were also observed in another group of 11 paired cord and infant specimens. Erythrocyte T3 uptake for the mothers was 11.3 per cent, which is significantly lower than that of normal adults, corroborating Hamolsky's~ observations. Parallel determinations of BEI are recorded for 5 of the 7 sets of infants studied in this special group. It will be noted from the values shown in the table that the BEI's for the cord bloods are high but distinctly less than the maternal values, while there is again an increase at 3 to 5 days. Thus, the relationship of the BEI to per cent T3 uptake in maternal blood differs from that in infant cord blood. Premature infants were then compared with full-term infants. Eleven healthy prematures 3 to 9 days of age showed a mean uptake of 26.5 per cent, compared to a mean of 19.8 per cent for full-term infants of a
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N e o n a t a l thyroid [ u n c t i o n
similar age. Nine prematures, 2 to 4 weeks of age, had a m e a n of 25.0 per cent, as opposed to 17.5 per cent for the full-term infants of the same age. T h e m e a n for prem a t u r e infants 6 to 9 weeks of age, 23.3 per cent, differs from the mean of 13.2 per cent for full-term infants of the same age. T h e differences between all three pairs are significant; the probability of this being due to chance is less than 0.01. Thus, the level of uptake in premature infants is higher than the level of uptake in full-term infants at the same age (Table V I and Fig. 4). Butanol extractable iodine values in 5 premature newborn infants were not higher than those in full-term infants.
Table IV. Per cent erythrocyte Ts uptake of full-term infants Significance
N o . o'f
sub-
Mean
4"
standard
Comparison with preceding age group
Comparison with "'normal" children [rom 11/2 to 15 years
Age
jects
error
Cord 3 to9 days 2 to4 weeks 6 to9 weeks
19
16.3+-0.7
56
19.8+0.5
p < 0.01
p ~ 0.01
18
17.5 +-0.7
p ~ 0.05
p < 0,01
24
13.2 +0.8
p ~ 0.01
p ~ 0.1
--
p ~ 0.01
DISCUSSION Hamolsky 7 has offered the following explanation of the mechanism of the test. Although several plasma protein elements bind thyroid hormone, it is believed that one, an alpha globulin, binds material more firmly than the others. E r y t h r o c y t e Ts uptake is apparently directly related to the degree of saturation of alpha-globulin binding sites by thyroxin-like material. W h e t h e r the effect noted is a specific competitive relationship between the red cell and the plasma binding sites, or whether this p h e n o m e n o n is a result of saturation and overflow is as yet somewhat uncertain. I n any event, for practical
3 5
Fig. 2. S E M ~- Standard error of the mean. Dotted lines represent 2 standard errors above and below the mean for 40 "normal" children, 11/2 to 15 years of age.
purposes, it could be postulated that in hyperthyroidism m o r e of the presumed binding sites would be occupied so that the added triiodothyronine would be attached to the more loosely b o u n d sites, and thence to the surface of the red cells producing the elevated erythrocyte Ta uptake. T h e converse would occur with hypothyroidism. A l t h o u g h triiodothyronine uptake on the red cell usually parallels other parameters of thyroid function, there are situations where the protein-bound iodine is elevated and the triiodothyronine uptake is depressed. An example of this is found in pregnancy, where there is a p r i m a r y increase in thyroidbinding globulin and an elevated circulating PBI secondary to the increased binding capacity. I n g b a r 9 has shown that thyroid-binding protein is greatly increased during pregn a n c y and to a lesser but significant degree in cord plasma. Thus, red blood cell T~
Table V. Per cent erythrocyte Ta uptake in a paired series of mothers and their infants
T3 Uptake
Significance
Mother 11.3 + 0.4 -Cord 14.8+0.9 p ~ 0 . 0 1 Infant 21.3+0.9 p ~ 0 . 0 1
BEI (meg.~ 100 ml.)
Significance
8.1 + 0.4 -5.6+0.5 p ~ 0 . 0 1 9.1+0.7 p ~ 0 . 0 1
3 6 Marks, Wolfson, and Klein
January 1961
Table VI. Per cent erythrocyte T3 uptake of full-term and premature infants
age 3 to 9 days 2 to 4 weeks 6 to 9 weeks
I
FuZZ-ter,~infant, 19.8_+0.5 (56) e 17.5+0.7 (18) 13.2 +0.8 (24)
I
eremature infants 26.5+1.2 ( l l ) t 25 +1.6 (9) 23.3+- 1.9 (5)
l
Signi~can. p<0.01 p<0.01 p < 0.01
"~Number of subjects in parentheses. t T h e m e a n B E I for 5 of these p r e m a t u r e newborn infants is 6.5 meg. per 100 ml. with a S.E.M. of • 0.3.
uptake probably reflects two factors: (1) the level of circulating thyroid-binding protein, and (2) its relative level of saturation by circulating thyroid hormone. One could postulate that in a specimen with a low hematocrit the larger plasma volume would contain relatively more thyroidbinding protein and consequently erythrocyte T3 uptake values would be falsely low. Our results showing an increase in erythrocyte Ta uptake parallel to an increase in hematocrit afford some evidence that this m a y be so. Other explanations are possible, however. Among these was one suggested by the work of Meade, 1~ who reported that erythrocyte T3 uptake was inversely proportional to the amount of washing of the red cells. However, we were unable to find any evidence to confirm this possibility in the single experiment carried out with different volumes of saline washes. Because of the wide variation in hematocrit seen in early infancy, all assays in the body of this report were carried out by the modification of Hamolsky's technique with the hematocrit adjusted to within the adult range (from 42 to 50 per cent) at the start of the procedure. When Danowski 1 first reported on his findings in 1951, he discussed several different possible explanations of the elevated protein-bound iodine noted in the first year of life: (1) that this represented a true increase in thyroid function, and (2) that the findings could be explained by changes in extracelIular fluids usually seen in newborn infants. Danowski discarded the latter theory, however, because this would not explain the persistence of elevation throughout as long a period as he demonstrated. In addition, the magnitude of changes in fluid volume are
not great enough to explain the elevations of protein-bound iodine. Danowski also raised the possibility that the infant metabolized thyroid hormones at a slower rate. This seems unlikely, however, in view of the work of Stevenson, 11 who showed that premature infants could ingest 120 to 180 rag. thyroid daily without clinically manifest toxicity. Another explanation for the elevated PBI or BEI, not discussed by Danowski, is in terms of some hormonally inert iodinated protein. At the present time, however, investigation of this possibility is not feasible since it would require the in vivo use of radioisotopes in newborn infants. Danowski gives two other possibilities: (1) that the elevated PBI is secondary to an increased plasma thyroid-binding capacity, and (2) that it is a reflection of transplacentally transfused material. I t will be noted that there is a difference between the triiodothyronine uptake and the protein-bound iodine in the newborn as corn-
Fig. 3. Mean + standard error for per cent T~ uptake shown at bottom of graph.
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N e o n a t a l thyroid f u n c t i o n
% R B C T~. U P T A K E IN T E R M AND PREMATURE INFANTS
3 7
of normal for full-term newborn infants, suggests that the thyroid-binding protein is less elevated in the premature than in the fullterm infant.
:30-
25
SUMMARY 2o
- - 2 SEM
+2SEM
15
B,
, ~ -~:a PREMATURE FULL T E R M
--2
I0 AGE-
Fig. 4. S E M
i
=
5 - 9 DAYS
z
2-4
WEEKS
-MEAN
SEM i
6~9 WEEKS
Standard error of the mean.
pared with that of the mother. I n the maternal serum, the PBI or B E I is elevated secondarily to an increased thyroid-binding protein as was previously demonstrated by Ingbar. 9 Both our findings and those of Hamolsky, 6 however, show a depression of the per cent T3 erythrocyte u p t a k e in maternal blood. I n addition, observations show that the cord blood obtained from the infants of the same mothers has a somewhat smaller BEI but an erythrocyte Ta uptake which is significantly greater both than that of the mothers and that of normal older children or adults. These changes show a significant discrepancy between the normal and umbilical cord findings. Thus, it is strongly suggested that the changes observed are more than a reflection of maternal thyroid status in the infant. T h e finding of an elevated erythrocyte T3 uptake in the newborn makes it seem impossible to explain the concurrently elevated PBI or B E I solely on the basis of an elevated thyroid-binding protein. If the increased PBI were secondary to an increased thyroid-binding capacity, one would expect a low T3 uptake and a high PBI, as seen in pregnancy. O u r findings, coupled with those of Danowski and of V a n Middelsworth, strongly suggest that there is a true increase in thyroid function in the newborn period. T h e finding of a more elevated Ta uptake in the premature, who has a BEI within the range
1. T h e erythrocyte Ta uptake method of Hamolsky has been modified so that it is adaptable to individuals in the pediatric age range. 2. Using this technique, we have shown elevated erythrocyte Ta values in the fullterm neonate. These fall to within the adult range when the infant is 6 to 9 weeks of age. T h e elevation of erythrocyte Ta uptake appears more m a r k e d and more prolonged in the premature infant. 3. T h e elevated erythrocyte Ta uptake shows that the increased protein-bound iodine o r butanol extractable iodine of the neonate is not the result of an increased thyroid-binding protein. T h e different relationships of the Ta uptake to the BEI in the maternal and in the cord blood specimens indicate t h a t the observations in the newborn are not merely a reflection of maternal thyroid status. O u r d a t a suggest that there is a true increase in thyroid secretion in the newborn infant. REFERENCES 1. Danowski, T. S., Johnson, S. Y., Price, W. C., MeKelvey, M., Stevenson, S. S., and McCluskey, E. R.: Protein Bound Iodine in Infants From Birth to One Year of Age, Pediatrics 7: 240, 1951. 2. Man, E. B., Picketing, D. E., Walker, J., and Cooke, R. E.: Butanol Extractable Iodine in the Serum of Infants, Pediatrics 9: 32, 1952. 3. Van Middelsworth, L.: Radioactive Iodine Uptake of Normal Newborn Infants, A. M. A. Am. J. Dis. Child. 88: 439, 1954. 4. Werner, S. C., Block, R. J., Mandl, R. H., and Kassenaar, A. A. H.: Pathogenesis of a Case of Congenital Goitre With Abnormally High Levels of SPI and With Mo.no and Diiodotyrosine in the Serum, J. Clin. Endocrinol. 17: 817, I957. 5. Stanbury, J. B., and Hedge, A. N.: A Study of a Family of Goitrous Cretins, J. Clin. Endocrinol. 10: 1471, 1950. 6. Hamolsky, M. W., Stein, M., and Freedburg, A. S.: The Thyroid Hormone-Plasma Protein
38
Marks, Wol[son, and Klein
Complex in Man, II. A New in Vitro Method for Study of "Uptake" of Labeled Hormonal Components by Human Erythrocytes, J. Clin. Endocrinol. 17: 33, 1957. 7. Hamolsky, M. W., Godoletz, A., and Freedberg, A. S.: The Plasma Protein-Thyroid Hormone Complex in Man, III. Further Studies of the in Vitro Red Blood Cell Uptake of Ilal-l-Triiodothyronine as a Diagnostic Test of Thyroid Function, J. Clin. Endocrinol. 19: 103, 1959. 8. Crigler, J. F., Hertz, J., and Hamolsky, M. W.: In Vitro Red Blood Cell Uptake of I13M-Triiodothyronine as a Measuremen.t of
January 1961
Thyroid Function in Children, A. M. A. J. Dis. Child. 98: 665, 1959. 9. DoMing, J. T., Freinkel, N., and Ingbar, S. H.: Thyroxin-Binding by Sera of Pregnant Women, Newborn Infants and Women With Spontaneous Abortion, J. Clin. Invest. 35: 1263, 1956. 10. Meade, R. C.: Possible Errors in Determination of RBC Uptake of 1131 Triiodothyronine, J. Clin. Endocrinol. 20: 480, 1960. 11. Stevenson, S. S., Wirth, P., Bastiani, R., and Danowski, T. S.: Some Effects of Exogenous Thyroid or Thyroxine Upon Premature Infants, Pediatrics 12: 263, 1953.
A strange cause of death English newspapers have recently reported the death of a 9-year-old boy caused by inhaling gasoline fumes. For some time the parents had observed the strange attraction of the boy [or gasoline fumes and had reprimanded and punished him. On the fatal day, while his father was working nearby, the boy crept under a canvas sheet which covered his father's motorcycle and removed the cap from the gasoline tank. After being missed [or three hours he was discovered dead with his face beside the open gasoline tank.