Changes in plasma thyroid hormone concentrations in chronically food-restricted female rats and their offspring during suckling.

To examine the joint effects of lactation and dietary restriction on thyroid function, we studied chronically food-restricted rat dams at conception and dams and their pups during the nursing period compared with controls that had free access to food. As expected, both dietary restriction [plasma thyroxine and 3,3',5-triiodothyronine (T3) values were lower and reverse T3 (rT3) values were higher in food-restricted than in control rats] and stage of lactation (T3 values decreased in both groups) had independent effects on maternal thyroid function. There also were interactive effects: rT3 values decreased during lactation among the food-restricted dams but remained constant among controls. Maternal dietary restriction also did not have a consistent effect on pup thyroid function during the nursing period: in offspring of food-restricted compared with control dams, plasma thyroxine values were lower only at d 14, T3 values were lower at d 7-21, and rT3 values were higher from birth to d 14. These data demonstrate that dietary restriction and lactation have synergistic negative effects on thyroid status in lactating rats and their pups and that thyroid hormone ontogeny is retarded in the offspring of food-restricted rats. These hormonal effects may contribute to the impaired milk production of food-restricted rats and to altered growth characteristics of their offspring.

Effect of iopanoic acid on basal and thyrotropin-stimulated thyroid hormone levels in suckling rat pups.

We proposed that basal and thyrotropin (TSH)-stimulated thyroid hormone levels of rat pups would be altered in the presence of iopanoic acid (IA), a radiographic contrast agent which competitively inhibits T4-to-T3 conversion, and that the nature of these changes would further depend upon the route of TSH administration in a manner distinct from that reported in adults. To test this hypothesis, litters from 24 Sprague-Dawley female rats were adjusted to 8 pups each. On day 5, 80 pups received IA (2.5 mg/100 g body weight) injections. On day 8, control and IA pups were further subdivided, and given bovine TSH (bTSH) either by subcutaneous injection or by intragastric gavage (to simulate milk-borne TSH intake), and then sacrificed 0, 1.5, or 3 hours later. We found significantly higher T4 and reverse-T3 (rT3) levels in IA-treated pups, but IA had no effect on basal or TSH-stimulated T3 levels attained, regardless of route of bTSH administration or time post-treatment. Our data demonstrate that the effects of IA on T4 and rT3 levels in the immature rat are comparable to those observed in adult rats and humans, but that the marked depression of T3 levels found in IA-treated adults does not occur in the 8-day old rat pup. We speculate that the IA-treated suckling pup's ability to sustain normal basal T3 levels and generate elevated T3 concentrations in response to TSH stimulation may reflect the activity during development of a T4-5'-deiodinase relatively resistant to competitive inhibition by this drug.

Suckling, but not formula feeding, induces a transient hyperthyroxinemia in rat pups.

To assess the effect of breast feeding on circulating thyroid hormone levels in the suckling neonate, we examined serum concentrations of T4, T3, and rT3 by RIA in 12- and 15-day-old rat pups fasted for 3 or 6 h before, at, and hourly for 3 or 4 h after suckling. In both age groups, serum T4 levels decreased significantly in response to the 6-h fast; no significant differences were observed after 3 h. T3 levels in 15-day-old (but not 12-day-old) rats were likewise significantly depressed, and rT3 levels were higher relative to those in the prefast group. After a 90-min suckling period, serum total and free T4 concentrations of 12-day-old pups increased 1 h postsuckling to levels significantly higher than those in the fasted animals; maximum levels were achieved at 2 h. T3 levels were unchanged over this period. In contrast, elevated total and free T4 and T3 levels and a reciprocal decrease in rT3 concentration, were evident immediately after the 90-min suckling period (0 h) in 15-day-old rat pups and increased through 1 h to levels significantly higher than those in the prefast group. After refeeding (0 h) of 3-h fasted pups, serum T4 levels increased significantly over fasted levels in suckled 12- and 15-day-old rats, as did T3 levels in 15-day-old pups, through 2 h postsuckling. In contrast, thyroid hormone levels of pups gavage-fed a commercial formula preparation at no time exceeded those of the prefast groups at either age, despite highly comparable stomach weights for both formula-fed and suckled pups at the time of refeeding. We conclude that 1) rat pups between the ages of 12 and 15 days postpartum become hypothyroxinemic between 3 and 6 h of milk deprivation, and 2) this condition is reversed by significant increases in T4 and T3, rendering the animal transiently hyperthyroxinemic after suckling, but not in response to formula feeding. Our data further demonstrate the critical nature of study designs involving fasts (either planned or incidental) of suckling rats in light of the observed pronounced variations in their thyroid hormone status relative to time postsuckling.

Developmental changes in rat thyroid responsiveness to thyrotropin administered by the subcutaneous and peroral route.

It has been demonstrated that orally administered thyrotropin (bovine, bTSH) evokes an increase in circulating T4 and T3 levels in 15-day-old suckling rat pups, but not in weaned animals. Because the feedback mechanisms of the hypothalamo-pituitary-thyroid axis change dramatically during the neonatal period, we chose to examine the efficacy of exogenous bTSH in eliciting a thyrostimulatory response via the subcutaneous (sc) or peroral (po) route in rat pups at 5, 8, 12, and 15 days postpartum. Suckling pups were divided into four groups and received one of the following: (i) 2 IU bTSH/100 g body wt administered sc; (ii) distilled H2O (dH2O) sc; (iii) 2 IU bTSH/100 g body wt given po; (iv) dH2O po. Animals were sacrificed at Time 0 and 1, 2, and 3 hr post-treatment, and the collected serum was analyzed for T4 and T3 by RIA. Maximum serum T4 levels were attained at 2-3 hr post-treatment, and the T4 response to sc-bTSH was significantly greater than that of the po-bTSH groups at all ages examined. This difference became progressively greater with increasing age, due to a persistent decline in T4 responsiveness in animals receiving po-bTSH. No significant differences in T4 or T3 levels attained were observed in 8-day-old rat pups treated with rat vs bovine TSH, either sc or po. Percentage T4 response (vs basal levels) steadily declined between Days 5 and 15 postpartum, in both sc- and po-bTSH treatment groups. Percentage T3 responsiveness to sc-bTSH also declined between 5 and 12 days postpartum, after which time T3 generation increased. Our results suggest that the neonatal rat is highly responsive to exogenous TSH late in the first week of life, and that the permeability of the gut at this stage of development further facilitates the impact of orally ingested TSH in the suckling.

Thyroid hormone-binding inhibitor in normal, pregnant, and lactating rat and postmenopausal human breast tissue.

The regulatory role of thyroid hormones in the synthesis of milk proteins and functional differentiation of the murine mammary gland has been demonstrated. Further, T4 and T3 and their metabolites are found in both rat and human milk. Since the enhanced metabolic demands of mammo- and lactogenesis may result in increased local hormonal requirements, we examined breast tissue from normal (virgin), pregnant, and lactating female rats, as well as postmenopausal human breast tissue for the presence of a thyroid hormone-binding inhibitor (THBI). This substance, which decreases binding of T4 to serum T4-binding globulin, has been described in rat liver, kidney, muscle, and intestine, and in the circulation of patients with nonthyroidal illnesses. THBI activity in the present study was assessed by equilibrium dialysis in whole tissue homogenates; the ether-soluble fraction was also analyzed for THBI activity by RIA. In a range of 0.8-8.0 mg tissue protein, we found lactating rat breast to contain elevated THBI levels when compared to those found in normal, virgin breast tissue. Half-maximum binding inhibition was achieved at 0.15 mg lactating breast tissue protein, vs. 0.23 mg breast tissue protein and 0.35 mg liver protein in homogenates from normal resting animals. Expressed in tissue milligram equivalents, half-maximum binding inhibition of lactating, 18-day pregnant, and normal rat breast tissue was 0.7, 2.6, and 4.8 mgeq, respectively. Thus, the THBI activity of lactating rat breast tissue was more than 3-fold greater than that of the pregnant rat and 7-fold greater than that found in resting breast tissue. Binding inhibition in postmenopausal human breast tissue was comparable to that of rat breast and liver tissue at low protein concentrations, but the maximum inhibition attainable (205 +/- 6%, n = 3) was significantly lower than that achieved by normal rat breast tissue (338 +/- 6%, n = 6; means +/- SEM). The ether-soluble fraction of breast tissue homogenates was also analyzed employing a THBI-RIA. THBI activity, expressed as percent binding inhibition, was elevated in extracts from lactating rat breast tissue in comparison to tissue from pregnant or normal animals at 6.0, 12.5, and 25 mgeq; the largest differences were observed at 25 mgeq tissue: lactating (57.8 +/- 2.3%) vs. pregnant (46.5 +/- 3.1%, P less than 0.01) vs. normal (41.7 +/- 2.3%, P greater than 0.05); n = 3 in all cases.(ABSTRACT TRUNCATED AT 400 WORDS)

Thyroxine-binding globulin in serum and milk specimens from puerperal lactating women.

The whey fraction of human milk contains a thyroxine-binding protein analogous to serum thyroxine-binding globulin. To assess the method by which this binding protein appears in milk over the course of lactation, paired serum and milk specimens from 45 healthy, euthyroid women were assayed for thyroxine-binding globulin and whey thyroxine-binding globulin using a highly specific thyroxine-binding globulin-radioimmunoassay. Elevated serum levels (39.2 +/- 4.72 micrograms thyroxine-binding globulin/mL; mean + SD, N = 5) were found in samples drawn at less than two weeks post partum. These levels normalized rapidly over the next three weeks post partum (26.0 +/- 3.3 micrograms/mL, P less than .005, N = 5), and by five weeks, no further significant decreases were observed. As was the case for serum thyroxine-binding globulin, whey thyroxine-binding globulin was present in its highest concentrations (79.3 +/- 13.4 micrograms/dL, N = 7) in the colostrum and transitional milk. These levels are equivalent to approximately 2 to 3% of serum concentrations. In contrast to serum thyroxine-binding globulin levels, however, whey thyroxine-binding globulin declined logarithmically over a protracted 12-week period, and no further significant changes in whey thyroxine-binding globulin concentrations were observed up to 20 weeks post partum, at which time they were approximately equal to 1% of serum thyroxine-binding globulin levels. A strong linear correlation between serum and whey thyroxine-binding globulin was found within the first five weeks post partum (r2 = .95, P less than .0005, N = 14).(ABSTRACT TRUNCATED AT 250 WORDS)

A thyroxine-binding protein in human breast milk similar to serum thyroxine-binding globulin.

A thyroid hormone-binding substance (TBGw) similar to serum T4-binding globulin (TBG) has been identified in the whey fraction of human breast milk. TBGw coeluted with serum TBG, as determined by Bio-Gel P-100 chromatography, and has an isoelectric point of 4.2-4.8, similar to that of serum TBG. The affinity constant of TBGw for T4 was similar to that of serum TBG(Ka, 1.54 +/- 0.38 X 10(9) M-1). Marked inhibition of TBGw binding of [125I]T4 was achieved by the addition of 1.5 X 10(-3) M 8-anilino-1-naphthalene-sulfonic acid. An albumin-like low affinity site (Ka, greater than 10(7) M-1) was also found. RIA of whey concentrates serially diluted in TBG-depleted serum indicated nearly identical binding curves for TBGw and TBG, with slopes of 2.33 (n = 15; r = 0.947) and 2.54 (n = 7; r = 0.996), respectively. Using a specific TBG RIA, a TBGw concentration of 0.29 +/- 0.08 microgram/ml (mean +/- SD) in breast milk (n = 26) was determined. Paired serum and whey specimens from individuals between 6 and 20 weeks of lactation were analyzed by TBG RIA; in these individuals, mean serum TBG concentrations were 25.0 +/- 3.4 micrograms/ml (n = 7); corresponding TBGw levels were 0.26 +/- 0.08 micrograms/ml (n = 7), i.e. approximately 1% of serum levels. Nonetheless, linear regression analysis of the data revealed no significant correlation between serum and whey TBG concentrations in these individuals.

Separation and radioimmunoassay of T3 and T4 in human breast milk.

There is little agreement among published reports of the radioimmunoassayable thyroid hormone content of breast milk, likely due to wide variations in methodology applied. In order to achieve a higher degree of specificity in the determination of T3 and T4 concentrations in breast milk, samples were ethanol-extracted and then chromatographed on an LH-20 column. Using this method, all T3 and T4 RIA activity eluted with the void volume. Following pancreatin digestion and subsequent extraction of whole milk samples, void volume T3 RIA activity decreased, and T3 co-eluted primarily with a standard preparation of T3 or 125I-T3, at a concentration of 275 +/- 132 ng/dl (mean +/- SD) (n = 9). In contrast, the elution volume of T4 RIA activity appeared unaffected by pancreatin. These data indicate that immunoreactive T3 and T4 are differentially bound to a thyroid hormone 'binding' substance present in breast milk. They further support the hypothesis that thyroid hormone sufficient to supplement the thyroid economy of the thyroid-deficient suckling infant is present in human breast milk.

n-acetyl-beta-hexosaminidase activity in human breast milk.

1. The lysosomal enzyme, N-acetyl-beta-hexosaminidase (HEX) is present in human breast milk. It is composed predominantly of "A" (heat-labile) and "B" (heat-stable) isozymes which coelute with the corresponding major serum isozymes on DE-52 ion-exchange chromatography. 2. Total HEX activity in "early" milk obtained at 2.8 +/- 1.4 weeks post partum, is approx. 2.5-fold higher (87 +/- 29 nmol/60'/mg protein. n = 10) than that of pregnancy serum (35.7 nmol/60'/mg protein) prior to delivery. 3. These levels increase to greater than 3-fold (110 +/- 20 nmol/60'/mg protein, n = 13) as the milk matures (10.3 +/- 4.2 weeks). 4. The specific activity of HEX A in the milk changes little with time post partum, because absolute 5. In contrast, HEX B specific activity is increased, as absolute levels (per volume) remain constant in the face of decreasing milk protein content, 5. These changes result in a high degree of correlation (r = 0.81) between time of lactation and % HEX A observed.

Thyrotropin in human breast milk.

The thyrotropin (TSH) content of human breast milk was investigated, using both direct 125I-TSH radioimmunoassay and radioimmunoassay preceded by a partial purification step of elution from concanavalin A-Sepharose 4B. 15 samples of human milk between 1 and 13 weeks post-partum were found to contain 2.0 +/- (SD) 0.9 microU TSH/ml when assayed after Con A-extraction and adjusted for recovery by interpolation of a standard curve of Con A-extracted TSH standards. Recovery of 125I-hTSH tracer from both serum standards (58.6 +/- 5.5%; n = 10) and milk (53.6 +/- 6.7%; n = 15) were comparable. TSH levels obtained post-extraction were not significantly different from those that TSH is present in human breast milk in low concentrations, comparable to those normally found in the serum of euthyroid adults.

Changes in serum N-acetyl-beta-hexosaminidase levels after treatment of hypothyroid and hyperthyroid individuals with L-thyroxine and propylthiouracil.

Multiple serum samples were obtained from six hypothyroid and six hyperthyroid females, 11--17 years of age, over the course of their corrective treatment with L-thyroxine (LT4) and propylthiouracil (PTU), respectively. Sera were assayed for total N-acetyl-beta-hexosaminidase (HEX), the A (heat-labile) and B (heat-stable) isozymes, and total T4. HEX activity (total HEX A) in sera from hypothyroid (< 4 micrograms/dl T4) patients (total HEX: 518 +/- 66 nmol/60 min/ml, mean +/- S.D.; HEX A: 325 +/- 55; n = 5) was significantly lower than that of the euthyroid control group (total HEX: 638 +/- 77 (p < 0.005); HEX A: 420 +/- 76 ( p < 0.01); n = 23); no difference in HEX B levels was noted. Serum samples from patients successfully treated for hypothyroidism via oral administration of LT4 (n = 12) displayed levels of total HEX (722 +/- 113) and HEX A (491 +/- 91) significantly higher than those of the control group (p < 0.01 in both cases); again, no change in levels of HEX B was observed. HEX activity in sera from hyperthyroid (> 13 micrograms/dl T4) individuals (total HEX: 839 +/- 96; HEX A: 540 +/- 74; HEX B: 299 +/- 52; n =20) was significantly higher than that of the control group (p < 0.005 in all cases). The depression of hormone activity to the euthyroid range by PTU was accompanied ay a decrease in enzyme activity to control levels (total HEX: 632 +/- 92; HEX A: 400 +/- 55; HEX B: 232 +/- 52; n = 16). Non-parametric analysis of the data shows highly significance differences between pre- and post-treatment enzyme levels (alpha < 0.001) in both hyper- and hypothyroid groups. Alteration of thyroid status, and specifically T4 level is, therefore, indicated to be a contributing factor in the regulation of serum HEX activity in humans, as evidenced by individual responsiveness to oral administration of this hormone, or inhibitors of its peripheral metabolism.

Sex differences in serum levels of N-acetyl-beta-hexosaminidase in infancy: correlation of enzyme activity with testosterone levels.

Serum levels of N-acetyl-beta-hexosaminidase (HEX) (EC 3.2.1.30) activity in infants display a sexual dimorphism. Total HEX activity in males between 1 and 3 months of age is significantly elevated over female levels (male (M), 1535 +/- 300 nmol/60 min/ml; female (F), 1150 +/- 203, P < 0.0005), and the A (labile) isozyme constitutes a significantly lower proportion of the total activity present (M, 56.0 +/- 4.2, N = 24; F, 64.3 +/- 4.6, N = 21, P < 0.0005). These findings led us to investigate the relationship between testosterone concentration and HEX activity in serum. Samples from male (N = 36) and female (N = 33) infants between 1 and 6 months of age were included. In both sexes, a high degree of correlation (P < 0.0005) was observed between testosterone and total HEX (M, r = 0.71; F, r = 0.73), HEX A (M, r = 0.68; F, r = 0.56) and HEX B (M, r = 0.68; F, r = 0.72). An inverse relationship exists between testosterone levels and % A: M, r = -0.56; F, r = -0.38 (P < 0.0025 and 0.025, respectively). In contrast, no correlation between HEX levels and testosterone was evident in either male or female adults (r = 0.20 and 0.18, respectively). These data implicate testosterone in the regulation of HEX activity during the early months of human development.

Effects of cortisone and thyroxine on suckling rat liver N-acetyl-beta-glucosaminidase isozymes. Comparison with adult reactivity.

Suckling rat liver N-acetyl-beta-glucosaminidase (hexosaminidase) activity undergoes considerable fluctuation during the first two weeks of life. As two major forms of hexosaminidase (A, heat-labile, and B, heat-stable) are known to exist in both human and adult rat liver, we choose to examine the effect of the maturative hormones, thyroxine and cortisone, upon these isozymes during the suckling period. Between days 7 and 15, the observed developmental change is attributable solely to an increase in the 'A-like' (heat-labile) form of the enzyme; an enhanced response is seen in thyroxine-injected 11-15-day old animals. The response may be considered 'age-independent', as adult animals react in the same manner. In contrast, cortisone-injected sucklings show a decrease in both A and B isozymes, while in adults no changes in total activity or isozyme distribution are evoked. The ratio of hexosaminidase A to hexosaminidase B in suckling rat liver appears to shift in favor of the labile (A) isozyme early in development.

Relationship of thyroid status and serum N-acetyl-beta-glucosaminidase isoenzyme activities in humans.

Serum from 28 hyperthyroid, hypothyroid, and euthyroid pre- or early puberty females was examined for N-acetyl-beta-glucosaminidase (HEX) activity. Total, isoenzyme A (labile), and isoenzyme B (stable) levels were determined for this enzyme. A high degree of correlation (r = 0.76; p less than 0.001) exists between total hexosaminidase activity and thyroid hormone levels (as reflected by the Free Thyroxine Index). Examining each isoenzyme individually, A is selectively enhanced (r = 0.84; p less than 0.0005) whereas B displays no significant change regardless of thyroid activity. In hyperthyroid individuals, levels of total hexosaminidase (730 +/- 67 units) (mean +/- S.D.) and isoenzyme A (516 +/- 46) were significantly higher than levels found in either the hypothyroid (total: 547 +/- 30; isoenzyme A: 352 +/- 31) or euthyroid (total: 620 +/- 81; isoenzyme A: 423 +/- 45) groups. However, no change was observed in levels of isoenzyme B among hypothyroid (195 +/- 19), euthyroid (197 +/- 43) and hyperthyroid (215 +/- 32) groups. These data substantiate our earlier findings in the rat, wherein thyroxine administration evoked a similar response in the liver. They are of particular interest in light of the deficiency of HEX A in variants of GM2 gangliosidosis.