3,3',5'-Triiodothyronine (Reverse T3) and 3,3',5-Triiodothyronine (T3) in Fetal and Adult Sheep: Studies of Metabolic Clearance Rates, Production Rates, Serum Binding, and Thyroidal Content Relative to Thyroxine. INDER J. CHOPRA, JOSEPH SACK, AND DELBERT A. FISHER Department of Medicine, UCLA Center for the Health Sciences, Los Angeles, California and Department of Pediatrics, UCLA-Harbor General Hospital, Torrance, California iodothyronines, it was estimated that whereas thyroidal secretion may account for nearly all of serum T3 (or PR-T3) in the fetus and about 50% of serum T3 in adults, it accounts for only about 3% of the serum rT3 (or PR-rT3) in both fetal and adult sheep. The results suggest a) that elevated serum rT3 in the fetus is due to its decreased clearance and increased production by mono-deiodination of T4, and b) that low serum T3 in the fetus is due to its increased clearance and decreased production by mono-deiodination of T4. In addition, on the basis of discordant changes in the production of T3 and rT3 from T4, it appears that there may exist two separate, apparently specific, iodothyronine deiodinating activities—one cleaving the iodine atom at the 5'-position and the other acting in the iodine atom at the 5-position of the T4 molecule; 5'-iodothyronine deiodinating activity is apparently reduced in the fetus. {Endocrinology 97: 1080, 1975)
ABSTRACT. To examine the mechanism(s) responsible for high serum concentration of 3,3',5'-triiodothyronine (reverse T3, rT3) and low serum concentration of 3,3',5-triiodothyronine (T3) in the fetus, we studied metabolic clearance rates (MCR) and production rates (PR) of rT3, T3, and thyroxine (T4) in adult nonpregnant sheep and sheep fetuses in utero. The mean fetal MCR-rT3 was significantly lower than that in adult sheep, and the mean fetal PR-rT3 significantly higher. The mean fetal MCR-T3 was higher than, and the mean fetal PR-T3 similar to that in adult sheep. The mean fetal MCR-T4 and PR-T4 were both significantly higher than the corresponding values in adult sheep. The ratios of mean PR-rT3 to PR-T4 (rT/T4) were similar in fetal and adult sheep. However, the ratio of mean PR-T3 to PR-T4 (T3/T4) in the fetal sheep was much lower than that in the adult sheep. The low fetal MCR-rT3 was not attributable to high serum binding of rT3. On the basis of the thyroidal content and kinetics of
R
ECENT studies indicate that 3,3',5'triiodothyronine (reverse T3, rT3) is a normal component of human serum and of thyroglobulin, and that thyroxine (T4) is an important source of rT3 in the human circulation (1). One intriguing observation in these earlier studies was that the serum concentration of rT3 is markedly elevated in human cord blood. This finding was particularly interesting since the serum concentration of 3,3',5-triiodothyronine (T3), known to derive mainly from T4 in man (2-6), is much lower in cord serum than in adult human serum (1,7-10). There is at present little informaReceived March 5, 1975. Supported in part by USPHS Grants AM 16155, HD 04270, and NIH Research Career Development Award 1K04, 70225. Reprints: Dr. Inder J. Chopra, Department of Medicine, UCLA Center for the Health Sciences, Los Angeles, California 90024.
tion regarding the mechanism(s) of the elevated serum rT3 in the newborn. Several aspects of thyroid hormone economy in sheep are apparently quite similar to those in man (11-14). Thus, just as in man, serum T3 concentrations are lower and serum T4 levels higher in fetal than in adult sheep (13,14). Pilot studies suggested that sheep may also be similar to man with regard to the metabolism of rT3; serum rT3 concentrations are higher in fetal sheep than in the adult sheep. The studies to be described were undertaken to gain some insight into the mechanism(s) of the elevated serum rT3 and decreased serum T3 in the fetus and newborn using the sheep model. Materials and Methods Sources of sera. To study serum concentrations of rT3, T3, and T4) blood was obtained from catheters placed surgically in the jugular veins of
1080
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REVERSE T, AND T, IN FETAL AND ADULT SHEEP
1081
assay of T4 and T3, the contamination of our radioactive rT3 with either radioactive T3 or radioactive T4 would be minimal. The effect of any radioiodide contaminating labeled iodothyronines on the kinetics of iodothyronines would be minimized by the technique of analysis of specimens {vide infra). After obtaining baseline blood samples for measurements of rT3, T3, and T4 concentrations, tracer doses (50-100 AiCi) of [125I]-rT3 and/pr [131I]-T3 or [131I]-T4 diluted in hypothyroid sheep serum, were injected into the fetus or the adult non-pregnant ewe. Blood samples were obtained from the animal (fetus Hormonal measurements. Serum concentrations or adult) under study at 5 min intervals until of rT3, T3, and T4 were measured by radioimmuno- 20 min; at 10-15 min intervals until 1 h; at 30 assays described previously (1,15,16). Since L- min intervals until 3 h; at 60-90 min intervals rT3 was not available at the time, rT3-binding anti- until 10 h, and at 4-8-h intervals thereafter serum was produced by immunization of rabbits for up to 96 h. Thyroid uptake of radioiodine with D,L-rT3 conjugated to human serum albumin liberated from breakdown of iodothyronines was (1). However, we have now tested the relative minimized by oral administration of potassium reactivity of L-rT3 and D,L-rT3 in our rT3 RIA; the perchlorate, 400 mg twice daily, to the adult ewe displacement curves obtained with the two being studied or to the mother when the fetus was under study. Aliquots of injected radioisomers were essentially superimposable. active iodothyronines, diluted in sheep serum, were kept in the refrigerator (4C) to serve as Kinetic studies. Healthy adult non-pregnant ewes reference standards; reference standards and (Columbia or Columbia-Suffolk cross) and 110- specimens were processed identically. Iodo135-day pregnant ewes were maintained on thyronines were extracted from 250 ul aliquots baled alfalfa and given free access to water. The of plasma and diluted standards using the butanolperipheral kinetics of various iodothyronines (T4, alkali wash procedure described by Fisher et al. T3, rT3) were studied in the non-pregnant (19); this method consistently allows recovery of adult animals and in the sheep fetuses in over 95% of organic iodide and less than Utero. Indwelling, exteriorized catheters were 0.5% of inorganic iodide. All radioactive samples placed in one jugular vein of the fetal sheep, and were counted to a statistical accuracy of ±2% in a in the jugular vein of the adult non-pregnant dual-channel gamma counter (Nuclear-Chicago). sheep. Beta ring labeled [131I]-L-T4 and [131I]-L-T3 The data on radioactive iodothyronine remaining (SA 80-100 mCi/mg in each case) were obtained in plasma were expressed as percent dose/1 from Industrial Nuclear, St. Louis, Mo.; chro- plasma. Metabolic clearance rates (MCR) were matographic studies revealed less than 5% con- calculated by the non-compartmental integral tamination of labeled T4 with T3 or iodide and approach as described by Oppenheimer et al. of labeled T3 with T4 or iodide. [125I]-L-rT3 (SA, (6,20). Production rates (PR) were calculated 350-690 mCi/mg), prepared by beta ring radio- as the product of the serum concentration («t,g/l) iodination of 3,3'-L-diiodothyronine (T2), was and respective MCR values. MCR and PR were obtained from Abbott Laboratories, North Chi- expressed per square meter (m2) body surface cago, Illinois. Under the conditions employed, area per day to allow comparison of results in fetal iodination of T2 in the 5-position was unlikely and adult animals. Surface areas were calculated (17); labeled rT3 and labeled T2 were the using the formula, surface area = K (weight)2'3 as main products of radioiodination and these were proposed by Dittmer and Altman (21); the value carefully separated by Mr. B. J. Green at Abbott of K (i.e., 8.3) was obtained from their Laboratories, North Chicago, 111. using a modifica- publication. tion of the Sephadex column chromatography of Lissitzky et al. (18). Since contamination of unlabeled T2 with T3 or T4 was less than 3% as Thyroid glands. Thyroid glands were obtained judged by its cross-reaction in the radioimmuno- from 4 fetal sheep and from 5 adult sheep. fetal, newborn, and maternal sheep; catheters were placed in the fetuses after uterotomy under local anaesthesia. Serum was obtained from 5 pairs of date-bred, 95-140-day pregnant sheep (Columbia or Columbia Suffolk cross) and their fetuses; from 12 fetuses varying in gestational age from 80-140 days (term is 145 days in sheep); from neonatal sheep periodically from the time of delivery by uterotomy at 95-130 days of gestational age until 4 days of postnatal life; from 6 sheep, 42 days of age; and from 5 sheep, 90 days of age.
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Enclo • 1975 Vol 97 • No 5
CHOPRA, SACK AND FISHER
1082 REVERSE
700ng/100 ml 400-
100n
T
3
I
J
'4
o
MOTHER FETUS
160
16
120ng/ 100 ml
12100 ml
80-I
8-
40-
4-
MOTHER FETUS
Thyroid glands were homogenized and hydrolyzed with Pronase (Calhiochem) under the conditions described by Inoue and Taurog (22), as applied in our earlier studies (23). The hydrolysates were extracted with 2 volumes of 95% ethanol and the extracted rT3, T4, and T3 measured by RIA as described previously (23, 24). Serum binding of iodothyronines. The binding of rT3, T3, and T4 by serum proteins of fetal and adult sheep was compared by measurement of the dialyzable fractions of these iodothyronines using modifications of equilibrium dialysis methods previously described for human and rabbit sera (8,25-27). The dialyzable fraction of each iodothyronine was measured by addition of predialyzed radioactive iodothyronine to serum diluted 1:10 (26), dialysis in plastic cells, and precipitation of the dialysate with magnesium chloride (25). As noted in previous studies (27), pilot studies indicated that the dialyzable fraction determined by using diluted serum and simple arithmetic correction for dilution is underestimated compared with the results obtained with undiluted serum. Therefore, a factor which may render the values obtained in diluted serum comparable to those in undiluted serum was determined for each iodothyronine by simultaneous measurements of whole and 1:10 diluted pooled serum from 7 sheep fetuses and from 5 adult sheep. The values were similar in fetal and adult sheep sera and were, therefore, averaged. The average correction factors so determined, were 1.55 for T4, 1.58 for T3, and 2.89 for rT3. These values were applied to individual test samples which were tested only in 1:10 dilution.
FlG. 1. Comparison of serum concentrations of rT3, T3, and T4 in 5 pairs of maternal and fetal sheep. Solid horizontal line depicts the mean value in each category of the data.
MOTHER FETUS
Results Serum rT3 concentration in fetal and newborn sheep. Figure 1 presents the data on serum concentrations of rT3, T3, and T4 in 5 pairs of fetal and maternal sheep. The mean (±SE) serum rT3 concentration of 379 ± 103 ng/100 ml in fetal sheep was much higher (P < 0.05) than that of 96 ± 20 ng/100 ml in adult sheep. The mean serum T4 of 9.1 ± 1.9 /ug/100 ml in the fetus was modestly, but not significantly, higher than the value of 6.6 ± 1 . 1 /xg/100 ml in adult sheep. In contrast, the mean fetal serum T3 of 16 ± 1.0 ng/100 ml was much lower (P < 0.001) than that of 71 ± 4.6 ng/100 ml in adult sheep. The relative concentrations for the various iodothyronines in fetal and adult sheep were similar to those in human cord serum and adult human serum (1). To determine whether the elevated serum rT3 in the fetus is a phenomenon occuring simply near term or whether it is present earlier in gestation, we measured serum rT3 values in 12 fetal sheep varying in gestational age from 80 days to near term (140 days) (Fig. 2). Although a consistent trend of increase in fetal serum rT3 was not observed, high serum rT3 values were observed even at 90-110 days of gestational life. These data suggest that elevated fetal serum rT3 levels may occur early in the last one-third of pregnancy. Figure 3 presents the data on serum rT3, T3, and T4 in 3 newborn sheep during the
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1083
REVERSE T, AND T, IN FETAL AND ADULT SHEEP first 6 hours of postnatal life. There were no consistent changes in serum rT3 concentrations during this period, but serum T 3 and T4 values increase during the first few hours of life. We next investigated the longer term changes in serum rT3 in lambs; these data are shown in Fig. 4. There was a gradual decrease in serum rT3 levels during the first 3 to 4 days of life, and at 42 and 90 days of age the serum concentrations were quite comparable to those of adult sheep. Metabolic clearance and production rates. To study the mechanisms responsible for high serum rT 3 and low serum T 3 levels in the fetus as compared with the adult, we studied the MCRs and/or PRs of rT3, T3, and T 4 in fetal and adult sheep. The results are summarized in Table I. The mean MCR-T4 was 2.52 L/M2/day in adult sheep and 3.85 L/M2/day in the fetuses. These values are significantly different (P < .05). The mean PR-T4 values were 146 and 335 /xg/M2/day in the adults and fetuses, respectively (P < 0.005). The mean value for MCR-rT3 was much lower in fetal than in adult sheep (18.8 vs 73.7 L/M2/day, P < 0.025), but since the mean fetal serum rT 3 was markedly higher than that in adult sheep (Table 1), the mean fetal PR-rT3, 102 jLtg/m2/day, was nearly three times greater than the mean adult value of 38 /u,g/M2/day (P < 0.001). The mean MCR-T3 was higher in fetal than in adult sheep (80 vs 42 L/M2/ day, P < 0.02). However, since the mean T 3 concentration in fetal sheep was low, the mean daily fetal PR-T3 was similar to that of adult animals.
1000 -i
I 9 800
600400200-
20
40 60 80 100 GESTATIONAL AGE (days)
120
140
FIG. 2. Senim rT3 concentration in fetal sheep of different gestational ages.
the mean dialyzable fractions of T3, T4, and rT3 were all higher in the serum of fetal sheep than in adult sheep. These results indicate that the greater protein-binding of 800
REVERSE T3
>jg/100 ml
Dialyzable fractions of serum rTz, T3, and T4 in sheep. Although the MCR values of T 3 and T4 were usually higher in fetal than in adult sheep, the MCR-rT3 was markedly lower. This discrepancy could be explained if fetal serum proteins bound more rT3 and less T 3 and T4 than did adult sheep serum proteins. It is evident in Table 2 that
0
1
2 3 4 HOURS AFTER BIRTH
FIG. 3. Serum concentrations of rT3, T3, and T4 in newborn sheep during first 6 hours after delivery by uterotomy.
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CHOPRA, SACK AND FISHER
Endo • 1975 Vol 97 • No 5
RANGE OF VALUES IN ADULT SHEEP
AGE (days) FlG. 4. Serum rT3 concentrations in lambs during first 90 days of life.
rT3 is not the explanation for the lower MCR of rT3 in the fetal sheep, and suggest that the relative differences in the MCRs of the various iodothyronines in fetal and adult sheep are due to mechanisms operational at the level of tissues.
these calculations that rT3 and T4 are secreted in the proportions in which they exist in thyroglobulin. These calculations suggest that thyroidal secretion which results in a mean serum T4 of 8.9 jitg/lOO ml in fetal serum (Table 1), adds only 15 ng/100 ml 2 Relative proportions of rT3 and T4 in the to rT3 in serum (or 2.8 yu,g/M /day to the PR of rT3); this amount of rT3 is only thyroid. To examine the contribution of about 2.7% of that actually present in serum. thyioidal secretion to the relatively increased Similarly, it was calculated that a similar small production rate of rT3 in the fetal sheep, proportion of 2.6% of the serum concentrawe studied the relative proportions of rT3 tion (or daily PR) of rT3 in the adult may and T4 in fetal and adult sheep thyroid derive from thyroidal secretion of rT3. glands (Table 3). The T4/rT3 weight ratio These analyses suggest that the majority of [mean ± SEM (no.)] of 108 ± 2.9 (4) in the rT3 produced daily in the fetal as well as fetal thyroid glands was significantly (P adult sheep must derive from extrathyroidal < 0.05) lower than that of 132 ± 8 . 7 (5) in metabolism of T . 4 the adult sheep thyroid glands. The thyroidal T4/T3 weight ratios were similar in Discussion fetal and adult sheep thyroid (Table 3). The present as well as previous studies Thyroidal contribution to serum concentra- indicate that the qualitative patterns of tion (or daily PR) of rT3. To quantify circulating T4 and T3 levels in fetal sheep the contribution of thyroidal rT3 secretion relative to adult sheep are similar to those to the PR-1T3 in fetal and adult sheep, we observed in man at comparable stages of estimated the approximate thyroidal contribu- life (8-14). Thus, the serum T3 concentration by using MCR-1T3, MCR-T4, serum T4, tion is much lower in fetal sheep during the and rT3/T4 ratio in the thyroglobulin in last one-third of gestation than in adult calculations similar to those described sheep, whereas the fetal serum T4 level previously for T3 (5); it was assumed in is either higher than or comparable to that
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REVERSE T, AND T, IN FETAL AND ADULT SHEEP
1085
serum is much higher than that in the adult sheep serum. Moreover, the serum rT3 level changes little in the first several hours of life and slowly decreases thereafter to reach
in adult sheep (Fig. 1, Table 1, ref. 12,13). The present study reveals another similarity in thyroid metabolism between sheep and man; the rT3 concentration in fetal sheep
TABLE 1. Serum concentrations, metabolic clearance rates and production rates of reverse T3, T3 and T4 in fetal and adult sheep
Iodothyronine
Sheep no.
Weight (kg)
Body surface area I (M2)
Serum concentration /Ltg/100 ml
Metabolic clearance rate Liters/ day
Liters/ M2/day
Production rate Atg/day
/u,g/M2/day
Fetal sheep Reverse T3 (rT3)
146 356 184 154
Mean ± SE
3.12 ± 0.50 944 184 154 Y
T3
Mean ± SE
2.04 2.46 3.37 1.58 2.36 ±0.38
944 146 356 184
T4
2.49 4.16 2.46 3.37
Mean ± SE
114.4 100.5 113.2 79.1
21.6 19.0 13.8 20.8
17.5 21.7 17.2 14.8
3.35 ± 0.45
18.81 ± 1.75
17.8 ± 1.43
101.8** ± 8.19
0.045
ABSTRACT. To examine the mechanism(s) responsible for high serum concentration of 3,3',5'-triiodothyronine (reverse T3, rT3) and low serum concentration of 3,3',5-triiodothyronine (T3) in the fetus, we studied metabolic clearance rates (MCR) and production rates (PR) of rT3, T3, and thyroxine (T4) in adult nonpregnant sheep and sheep fetuses in utero. The mean fetal MCR-rT3 was significantly lower than that in adult sheep, and the mean fetal PR-rT3 significantly higher. The mean fetal MCR-T3 was higher than, and the mean fetal PR-T3 similar to that in adult sheep. The mean fetal MCR-T4 and PR-T4 were both significantly higher than the corresponding values in adult sheep. The ratios of mean PR-rT3 to PR-T4 (rT/T4) were similar in fetal and adult sheep. However, the ratio of mean PR-T3 to PR-T4 (T3/T4) in the fetal sheep was much lower than that in the adult sheep. The low fetal MCR-rT3 was not attributable to high serum binding of rT3. On the basis of the thyroidal content and kinetics of
R
ECENT studies indicate that 3,3',5'triiodothyronine (reverse T3, rT3) is a normal component of human serum and of thyroglobulin, and that thyroxine (T4) is an important source of rT3 in the human circulation (1). One intriguing observation in these earlier studies was that the serum concentration of rT3 is markedly elevated in human cord blood. This finding was particularly interesting since the serum concentration of 3,3',5-triiodothyronine (T3), known to derive mainly from T4 in man (2-6), is much lower in cord serum than in adult human serum (1,7-10). There is at present little informaReceived March 5, 1975. Supported in part by USPHS Grants AM 16155, HD 04270, and NIH Research Career Development Award 1K04, 70225. Reprints: Dr. Inder J. Chopra, Department of Medicine, UCLA Center for the Health Sciences, Los Angeles, California 90024.
tion regarding the mechanism(s) of the elevated serum rT3 in the newborn. Several aspects of thyroid hormone economy in sheep are apparently quite similar to those in man (11-14). Thus, just as in man, serum T3 concentrations are lower and serum T4 levels higher in fetal than in adult sheep (13,14). Pilot studies suggested that sheep may also be similar to man with regard to the metabolism of rT3; serum rT3 concentrations are higher in fetal sheep than in the adult sheep. The studies to be described were undertaken to gain some insight into the mechanism(s) of the elevated serum rT3 and decreased serum T3 in the fetus and newborn using the sheep model. Materials and Methods Sources of sera. To study serum concentrations of rT3, T3, and T4) blood was obtained from catheters placed surgically in the jugular veins of
1080
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REVERSE T, AND T, IN FETAL AND ADULT SHEEP
1081
assay of T4 and T3, the contamination of our radioactive rT3 with either radioactive T3 or radioactive T4 would be minimal. The effect of any radioiodide contaminating labeled iodothyronines on the kinetics of iodothyronines would be minimized by the technique of analysis of specimens {vide infra). After obtaining baseline blood samples for measurements of rT3, T3, and T4 concentrations, tracer doses (50-100 AiCi) of [125I]-rT3 and/pr [131I]-T3 or [131I]-T4 diluted in hypothyroid sheep serum, were injected into the fetus or the adult non-pregnant ewe. Blood samples were obtained from the animal (fetus Hormonal measurements. Serum concentrations or adult) under study at 5 min intervals until of rT3, T3, and T4 were measured by radioimmuno- 20 min; at 10-15 min intervals until 1 h; at 30 assays described previously (1,15,16). Since L- min intervals until 3 h; at 60-90 min intervals rT3 was not available at the time, rT3-binding anti- until 10 h, and at 4-8-h intervals thereafter serum was produced by immunization of rabbits for up to 96 h. Thyroid uptake of radioiodine with D,L-rT3 conjugated to human serum albumin liberated from breakdown of iodothyronines was (1). However, we have now tested the relative minimized by oral administration of potassium reactivity of L-rT3 and D,L-rT3 in our rT3 RIA; the perchlorate, 400 mg twice daily, to the adult ewe displacement curves obtained with the two being studied or to the mother when the fetus was under study. Aliquots of injected radioisomers were essentially superimposable. active iodothyronines, diluted in sheep serum, were kept in the refrigerator (4C) to serve as Kinetic studies. Healthy adult non-pregnant ewes reference standards; reference standards and (Columbia or Columbia-Suffolk cross) and 110- specimens were processed identically. Iodo135-day pregnant ewes were maintained on thyronines were extracted from 250 ul aliquots baled alfalfa and given free access to water. The of plasma and diluted standards using the butanolperipheral kinetics of various iodothyronines (T4, alkali wash procedure described by Fisher et al. T3, rT3) were studied in the non-pregnant (19); this method consistently allows recovery of adult animals and in the sheep fetuses in over 95% of organic iodide and less than Utero. Indwelling, exteriorized catheters were 0.5% of inorganic iodide. All radioactive samples placed in one jugular vein of the fetal sheep, and were counted to a statistical accuracy of ±2% in a in the jugular vein of the adult non-pregnant dual-channel gamma counter (Nuclear-Chicago). sheep. Beta ring labeled [131I]-L-T4 and [131I]-L-T3 The data on radioactive iodothyronine remaining (SA 80-100 mCi/mg in each case) were obtained in plasma were expressed as percent dose/1 from Industrial Nuclear, St. Louis, Mo.; chro- plasma. Metabolic clearance rates (MCR) were matographic studies revealed less than 5% con- calculated by the non-compartmental integral tamination of labeled T4 with T3 or iodide and approach as described by Oppenheimer et al. of labeled T3 with T4 or iodide. [125I]-L-rT3 (SA, (6,20). Production rates (PR) were calculated 350-690 mCi/mg), prepared by beta ring radio- as the product of the serum concentration («t,g/l) iodination of 3,3'-L-diiodothyronine (T2), was and respective MCR values. MCR and PR were obtained from Abbott Laboratories, North Chi- expressed per square meter (m2) body surface cago, Illinois. Under the conditions employed, area per day to allow comparison of results in fetal iodination of T2 in the 5-position was unlikely and adult animals. Surface areas were calculated (17); labeled rT3 and labeled T2 were the using the formula, surface area = K (weight)2'3 as main products of radioiodination and these were proposed by Dittmer and Altman (21); the value carefully separated by Mr. B. J. Green at Abbott of K (i.e., 8.3) was obtained from their Laboratories, North Chicago, 111. using a modifica- publication. tion of the Sephadex column chromatography of Lissitzky et al. (18). Since contamination of unlabeled T2 with T3 or T4 was less than 3% as Thyroid glands. Thyroid glands were obtained judged by its cross-reaction in the radioimmuno- from 4 fetal sheep and from 5 adult sheep. fetal, newborn, and maternal sheep; catheters were placed in the fetuses after uterotomy under local anaesthesia. Serum was obtained from 5 pairs of date-bred, 95-140-day pregnant sheep (Columbia or Columbia Suffolk cross) and their fetuses; from 12 fetuses varying in gestational age from 80-140 days (term is 145 days in sheep); from neonatal sheep periodically from the time of delivery by uterotomy at 95-130 days of gestational age until 4 days of postnatal life; from 6 sheep, 42 days of age; and from 5 sheep, 90 days of age.
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Enclo • 1975 Vol 97 • No 5
CHOPRA, SACK AND FISHER
1082 REVERSE
700ng/100 ml 400-
100n
T
3
I
J
'4
o
MOTHER FETUS
160
16
120ng/ 100 ml
12100 ml
80-I
8-
40-
4-
MOTHER FETUS
Thyroid glands were homogenized and hydrolyzed with Pronase (Calhiochem) under the conditions described by Inoue and Taurog (22), as applied in our earlier studies (23). The hydrolysates were extracted with 2 volumes of 95% ethanol and the extracted rT3, T4, and T3 measured by RIA as described previously (23, 24). Serum binding of iodothyronines. The binding of rT3, T3, and T4 by serum proteins of fetal and adult sheep was compared by measurement of the dialyzable fractions of these iodothyronines using modifications of equilibrium dialysis methods previously described for human and rabbit sera (8,25-27). The dialyzable fraction of each iodothyronine was measured by addition of predialyzed radioactive iodothyronine to serum diluted 1:10 (26), dialysis in plastic cells, and precipitation of the dialysate with magnesium chloride (25). As noted in previous studies (27), pilot studies indicated that the dialyzable fraction determined by using diluted serum and simple arithmetic correction for dilution is underestimated compared with the results obtained with undiluted serum. Therefore, a factor which may render the values obtained in diluted serum comparable to those in undiluted serum was determined for each iodothyronine by simultaneous measurements of whole and 1:10 diluted pooled serum from 7 sheep fetuses and from 5 adult sheep. The values were similar in fetal and adult sheep sera and were, therefore, averaged. The average correction factors so determined, were 1.55 for T4, 1.58 for T3, and 2.89 for rT3. These values were applied to individual test samples which were tested only in 1:10 dilution.
FlG. 1. Comparison of serum concentrations of rT3, T3, and T4 in 5 pairs of maternal and fetal sheep. Solid horizontal line depicts the mean value in each category of the data.
MOTHER FETUS
Results Serum rT3 concentration in fetal and newborn sheep. Figure 1 presents the data on serum concentrations of rT3, T3, and T4 in 5 pairs of fetal and maternal sheep. The mean (±SE) serum rT3 concentration of 379 ± 103 ng/100 ml in fetal sheep was much higher (P < 0.05) than that of 96 ± 20 ng/100 ml in adult sheep. The mean serum T4 of 9.1 ± 1.9 /ug/100 ml in the fetus was modestly, but not significantly, higher than the value of 6.6 ± 1 . 1 /xg/100 ml in adult sheep. In contrast, the mean fetal serum T3 of 16 ± 1.0 ng/100 ml was much lower (P < 0.001) than that of 71 ± 4.6 ng/100 ml in adult sheep. The relative concentrations for the various iodothyronines in fetal and adult sheep were similar to those in human cord serum and adult human serum (1). To determine whether the elevated serum rT3 in the fetus is a phenomenon occuring simply near term or whether it is present earlier in gestation, we measured serum rT3 values in 12 fetal sheep varying in gestational age from 80 days to near term (140 days) (Fig. 2). Although a consistent trend of increase in fetal serum rT3 was not observed, high serum rT3 values were observed even at 90-110 days of gestational life. These data suggest that elevated fetal serum rT3 levels may occur early in the last one-third of pregnancy. Figure 3 presents the data on serum rT3, T3, and T4 in 3 newborn sheep during the
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1083
REVERSE T, AND T, IN FETAL AND ADULT SHEEP first 6 hours of postnatal life. There were no consistent changes in serum rT3 concentrations during this period, but serum T 3 and T4 values increase during the first few hours of life. We next investigated the longer term changes in serum rT3 in lambs; these data are shown in Fig. 4. There was a gradual decrease in serum rT3 levels during the first 3 to 4 days of life, and at 42 and 90 days of age the serum concentrations were quite comparable to those of adult sheep. Metabolic clearance and production rates. To study the mechanisms responsible for high serum rT 3 and low serum T 3 levels in the fetus as compared with the adult, we studied the MCRs and/or PRs of rT3, T3, and T 4 in fetal and adult sheep. The results are summarized in Table I. The mean MCR-T4 was 2.52 L/M2/day in adult sheep and 3.85 L/M2/day in the fetuses. These values are significantly different (P < .05). The mean PR-T4 values were 146 and 335 /xg/M2/day in the adults and fetuses, respectively (P < 0.005). The mean value for MCR-rT3 was much lower in fetal than in adult sheep (18.8 vs 73.7 L/M2/day, P < 0.025), but since the mean fetal serum rT 3 was markedly higher than that in adult sheep (Table 1), the mean fetal PR-rT3, 102 jLtg/m2/day, was nearly three times greater than the mean adult value of 38 /u,g/M2/day (P < 0.001). The mean MCR-T3 was higher in fetal than in adult sheep (80 vs 42 L/M2/ day, P < 0.02). However, since the mean T 3 concentration in fetal sheep was low, the mean daily fetal PR-T3 was similar to that of adult animals.
1000 -i
I 9 800
600400200-
20
40 60 80 100 GESTATIONAL AGE (days)
120
140
FIG. 2. Senim rT3 concentration in fetal sheep of different gestational ages.
the mean dialyzable fractions of T3, T4, and rT3 were all higher in the serum of fetal sheep than in adult sheep. These results indicate that the greater protein-binding of 800
REVERSE T3
>jg/100 ml
Dialyzable fractions of serum rTz, T3, and T4 in sheep. Although the MCR values of T 3 and T4 were usually higher in fetal than in adult sheep, the MCR-rT3 was markedly lower. This discrepancy could be explained if fetal serum proteins bound more rT3 and less T 3 and T4 than did adult sheep serum proteins. It is evident in Table 2 that
0
1
2 3 4 HOURS AFTER BIRTH
FIG. 3. Serum concentrations of rT3, T3, and T4 in newborn sheep during first 6 hours after delivery by uterotomy.
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CHOPRA, SACK AND FISHER
Endo • 1975 Vol 97 • No 5
RANGE OF VALUES IN ADULT SHEEP
AGE (days) FlG. 4. Serum rT3 concentrations in lambs during first 90 days of life.
rT3 is not the explanation for the lower MCR of rT3 in the fetal sheep, and suggest that the relative differences in the MCRs of the various iodothyronines in fetal and adult sheep are due to mechanisms operational at the level of tissues.
these calculations that rT3 and T4 are secreted in the proportions in which they exist in thyroglobulin. These calculations suggest that thyroidal secretion which results in a mean serum T4 of 8.9 jitg/lOO ml in fetal serum (Table 1), adds only 15 ng/100 ml 2 Relative proportions of rT3 and T4 in the to rT3 in serum (or 2.8 yu,g/M /day to the PR of rT3); this amount of rT3 is only thyroid. To examine the contribution of about 2.7% of that actually present in serum. thyioidal secretion to the relatively increased Similarly, it was calculated that a similar small production rate of rT3 in the fetal sheep, proportion of 2.6% of the serum concentrawe studied the relative proportions of rT3 tion (or daily PR) of rT3 in the adult may and T4 in fetal and adult sheep thyroid derive from thyroidal secretion of rT3. glands (Table 3). The T4/rT3 weight ratio These analyses suggest that the majority of [mean ± SEM (no.)] of 108 ± 2.9 (4) in the rT3 produced daily in the fetal as well as fetal thyroid glands was significantly (P adult sheep must derive from extrathyroidal < 0.05) lower than that of 132 ± 8 . 7 (5) in metabolism of T . 4 the adult sheep thyroid glands. The thyroidal T4/T3 weight ratios were similar in Discussion fetal and adult sheep thyroid (Table 3). The present as well as previous studies Thyroidal contribution to serum concentra- indicate that the qualitative patterns of tion (or daily PR) of rT3. To quantify circulating T4 and T3 levels in fetal sheep the contribution of thyroidal rT3 secretion relative to adult sheep are similar to those to the PR-1T3 in fetal and adult sheep, we observed in man at comparable stages of estimated the approximate thyroidal contribu- life (8-14). Thus, the serum T3 concentration by using MCR-1T3, MCR-T4, serum T4, tion is much lower in fetal sheep during the and rT3/T4 ratio in the thyroglobulin in last one-third of gestation than in adult calculations similar to those described sheep, whereas the fetal serum T4 level previously for T3 (5); it was assumed in is either higher than or comparable to that
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REVERSE T, AND T, IN FETAL AND ADULT SHEEP
1085
serum is much higher than that in the adult sheep serum. Moreover, the serum rT3 level changes little in the first several hours of life and slowly decreases thereafter to reach
in adult sheep (Fig. 1, Table 1, ref. 12,13). The present study reveals another similarity in thyroid metabolism between sheep and man; the rT3 concentration in fetal sheep
TABLE 1. Serum concentrations, metabolic clearance rates and production rates of reverse T3, T3 and T4 in fetal and adult sheep
Iodothyronine
Sheep no.
Weight (kg)
Body surface area I (M2)
Serum concentration /Ltg/100 ml
Metabolic clearance rate Liters/ day
Liters/ M2/day
Production rate Atg/day
/u,g/M2/day
Fetal sheep Reverse T3 (rT3)
146 356 184 154
Mean ± SE
3.12 ± 0.50 944 184 154 Y
T3
Mean ± SE
2.04 2.46 3.37 1.58 2.36 ±0.38
944 146 356 184
T4
2.49 4.16 2.46 3.37
Mean ± SE
114.4 100.5 113.2 79.1
21.6 19.0 13.8 20.8
17.5 21.7 17.2 14.8
3.35 ± 0.45
18.81 ± 1.75
17.8 ± 1.43
101.8** ± 8.19
0.045
