J.J.M. de Vijlder

Thyroid function in very preterm infants: influences of gestational age and disease

It is not known how immaturity and disease influence postnatal thyroid function in infants <30 wk of gestational age. We performed serial measurements of plasma thyroxine (T4), free T4 (FT4), triiodothyronine (T3), reverse T3 (rT3), TSH, and T4-binding globulin (TBG) in 100 infants of <30 wk of gestation, during the first 8 postnatal weeks, to investigate the influences of disease and gestational age on the time course of thyroid hormones. One hundred infants were divided twice into two groups: 1) in a group of 25-28 and of 28-30 wk of gestation; and 2) in a sick and a healthy group, with similar gestational ages. The time course of T4, FT4, T3, TSH, and TBG, but not rT3 differed significantly (p < 0.005) between the gestational age groups. T4 and FT4 decreased to levels below the cord blood value with a deeper FT4 nadir on d 7 in the youngest group. Disease decreased T4, FT4, T3, TSH, and TBG concentrations especially during the 1st wk after birth (p < 0.005). However, the FT4 nadir on d 7 was similar in sick and healthy infants. After 3 wk, T4, FT4, T3, and TBG were higher in the sick group compared with the healthy group. rT3 levels were not increased in sick infants. We conclude that the extent of the FT4 decrease after birth in infants of <30 wk gestation is mainly influenced by gestational age and probably reflects a transient depletion of thyroidal hormone reserves. rT3 cannot be used as a marker of nonthyroidal illness in very preterm infants.

The reaction between NAD+ and rabbit-muscle glyceraldehydephosphate dehydrogenase

Abstract 1. 1. The A 280 mμ : A 260 mμ ratio of NAD + -free rabbit-muscle glyceraldehydephosphate dehydrogenase was found to be 2.00–2.03. 2. 2. Ultracentrifugation showed that 4 moles of NAD + may be bound to the enzyme per mole (mol. wt. 145 000). The first two molecules are bound stoicheiometrically within the experimental error ( K D μ M), while the third and fourth molecules are bound with dissociaton constants of 4 and 35 μM, respectively. 3. 3. The first three molecules of NAD + bound to the enzyme cause the formation of a band at 360 mμ of about equal intensity for each molecule. The fourth molecule causes little further increase of absorption at 360 mμ. 4. 4. A plot of the rate of reduction of NAD + by glyceraldehyde in the presence of arsenate against the NAD + concentration shows a sharp break in the curve at 4 moles NAd + per mole enzyme. 5. 5. Stopped-flow experiments showed that when up to 1 mole of NAD + is added to the enzyme, maximum absorbance increase at 360 mμ is reached within 3 msec. This corresponds to a second-order reaction constant of more than 10 8 M −1 ·sec −1 . With 2 moles of NAD + per mole of enzyme, 81% of the reaction is over in 3 msec, and with 3 or more moles NAD + about 75%. The reaction requires about 1 sec for completion. 6. 6. Prior treatment with NAD + speeds the change of absorbance obtained with subsequent additions of NAD + . The final value obtained, however, is unaltered. 7. 7. For the muscle enzyme under our experimental conditions, a model in which the binding of 1 NAD + molecule to one protomer affects the conformation of a second protomer, either before or after binding with NAD + , appear more appropriate than the allosteric model, which requires that the binding of any one ligand molecule is intrinsically independent of the binding of any other.

Thyroxine administration to infants of less than 30 weeks gestational age decreases plasma tri-iodothyronine concentrations

OBJECTIVE To investigate the effect on thyroid hormone metabolism of the administration of thyroxine to very preterm infants. DESIGN AND METHODS Two hundred infants of less than 30 weeks gestation were enrolled into a randomized, double-blind, placebo-controlled trial. Thyroxine (T4) (at a fixed daily dose of 8 microg/kg birthweight) or placebo was started 12-24h after birth and discontinued 6 weeks later. Plasma concentrations of T4, tri-iodothyronine (T3), reverse T3 (rT3), TSH, and thyroxine-binding globulin were measured weekly during trial medication and 2 weeks thereafter. RESULTS The T4 and the placebo group each comprised 100 infants. Antenatal, perinatal, and postnatal clinical characteristics were comparable in both groups. T4 and rT3 were significantly increased in the T4 group. TSH concentrations were depressed in the T4 group and T3 was significantly decreased, probably as a result of TSH depression. The T4/T3 and T4/rT3 ratios differed significantly between the two study groups. CONCLUSIONS Daily T4 administration during the first 6 weeks after birth to infants of less than 30 weeks gestation prevents hypothyroxinemia, but decreases plasma T3 concentrations. Our finding possibly implies that very preterm infants should receive supplements of both T4 and T3.

Use of amiodarone during pregnancy

UNLABELLED Five cases are studied in which amiodarone (AM) was given during pregnancy, in two of them also during the breast feeding period, to estimate the risks for adverse effects. We measured the concentrations of AM and its major metabolite desethylamiodarone (DEA) in maternal plasma, cord plasma, infant plasma, placental tissue and breast milk and the thyroid hormones were measured in maternal and neonatal serum. Also, the neonates were examined for AM-associated adverse effects over a period varying from 8 months up to 5 years. We observed a limited maternal-fetal transfer of AM and DEA, while the concentration of DEA in placental tissue is relatively high. Considerable amounts of AM and DEA were present in breast milk. One infant appeared to be hypothyroid, detected by the neonatal thyroid screening. He was treated with triiodothyronine for weeks, until it was clear that the thyroid dysfunction was resolved. The other infants had normal screening results. No effect of the AM medication was observed on growth, liver function or cornea and skin. IN CONCLUSION although pregnancy and lactation are no absolute contraindications for use of AM, special precautions are necessary. It is unavoidable that in some cases the pregnant mother, and especially her infant, becomes hypothyroid. AM has to be administered in the lowest possible dose, and the maternal and neonatal thyroid function must be controlled as long as the exposure to AM lasts.

Binding and properties of NAD+ in glyceraldehydephosphate dehydrogenase from lobster-tail muscle

Abstract 1. 1. The extinction coefficient at 280 nm of charcoal-treated lobster-muscle glyceraldehydephosphate dehydrogenase is 0.80 cm2·mg−1. 2. 2. The A 280 nm :A 260 nm of NAD+-free enzyme was found to be 1.99. 3. 3. Equilibrium dialysis showed that 4 moles of NAD+ may be bound to the enzyme per mole (mol. wt. 145 000). The first two molecules are bound stoicheiometrically within the experimental error ( K D −9 M ), whereas the third and fourth molecules are bound with dissociation constants of respectively 6·10−7 M and 13·10−6 M. 4. 4. The first three NAD+ molecules contribute equally to the absorption at 360 nm, whereas the fourth makes little if any contribution. 5. 5. On mixing NAD+ with the enzyme, the maximum increase of absorption at 360 nm is reached within 3·5 msec, independently of the amount of NAD+ added. This corresponds to a second-order rate constant of more than 1·1010 M−1·sec−1. 6. 6. The reduction of acetyl phosphate by NADH, and its arsenolysis, catalysed by the enzyme, require NAD+. NAD+ has no effect on the esterase activity.

Function and role of NAD+ in mechanism of action of rabbit-muscle glyceraldehydephosphate dehydrogenase.

1. 1. NAD+ is required for the oxidation of NADH by 1,3-diphosphoglycerate or acetyl phosphate. 2. 2. The maximal rate of acetyl phosphate reduction by NADH or APADH is reached when about 3 moles NAD+ per mole enzyme are added. Larger amounts of NAD+ inhibit with a Ki (NADH as substrate) equal to 45 μM. 3. 3. The NAD-binding site with the lowest affinity for NAD+ appears to be the most active in the dehydrogenase reaction. Under optimal conditions the E-(NAD)3 complex is the catalytically active enzyme. 4. 4. The E-(NAD)3 complex appears also to be the form with maximal catalytic activity in the transferase reactions. 5. 5. NAD+ can be replaced by APAD+ in both the dehydrogenase and arsenolysis reactions.

Studies on the mechanism of inhibition of nuclear triiodothyronine binding by fatty acids

Studies were designed to elucidate the mechanism by which unsaturated fatty acids inhibit the binding of triiodothyronine (T3) ro rat liver nuclei. The possibility of a direct interaction between oleic acid and T3 was excluded by dialysis experiments. Oleic acid inhibits nuclear T3 binding in a strictly competitive manner. The K i value of oleic acid was approx. 104 times greater than that of T3. The inhibitory effect of oleic acid could be reversed by bovine serum albumin.

Binding of NAD+ to rabbit-muscle glyceraldehydephosphate dehydrogenase as studied by optical rotatory dispersion and circular dichroism.

1. 1. The circular-dichroism spectra of both charcoal-treated and NAD+-containing glyceraldehydephosphate dehydrogenase from rabbit muscle were determined from 200 mμ to 500 mμ. 2. 2. The difference in the optical rotatory dispersion of the holoenzyme and the apoenzyme was calculated from the circular-dichroism spectra by the Kronig-Kramers transformation. 3. 3. The change in the Moffitt-Yang parameter, b0, and the change in molecular ellipticity at 350 mμ of the enzyme were followed as a function of added NAD+. The curves obtained gave a linear increase up to 2 molecules of NAD+ per molecule of enzyme, and an intersection point at 3.0 molecules NAD+ per molecule enzyme. 4. 4. By following the change in molecular ellipticity at 330 mμ on addition of 3-acetylpyridine-adenine dinucleotide, a similar result was obtained as with NAD+. 5. 5. Circular dichroism curves with a maximum at 330 mμ were found both with the enzyme-NADH complex and the enzyme-NAD+ complex treated with iodoacetate. 6. 6. Since binding of the enzyme with NAD+ has no effect on the optical rotatory dispersion or the circular dichroism below 250 mμ, it is concluded that the apo- and holoenzyme do not differ in overall protein conformation. The changes found above 250 mμ are at least partly due to extrinsic bands of the NAD+. 7. 7. The broad band between 300 and 450 mμ in the circular-dichroism spectrum of holoenzyme is probably composed of at least two components: (i) the charge-transfer complex between NAD+ and the reactive -SH group of the protein; (ii) an interaction between the adenine moiety and the enzyme.

Effects of maternal thyroid status on thyroid hormones and growth in congenitally hypothyroid goat fetuses during the second half of gestation

Congenital hypothyroidism in Dutch goats is due to a thyroglobulin (TG) synthesis defect that is inherited in an autosomal recessive manner. Minute amounts of mutated TG messenger RNA are translated into glycosylated TG fragments that contain the N-terminal hormonogenic site and are able to form T4, albeit less efficiently. We analyzed the effects of maternal thyroid status on fetal plasma thyroid hormones and growth during the second half of gestation (E90–E150). Maternal hypothyroidism, present from midgestation, resulted in decreased brain and cerebellum weights of affected goitrous fetuses, most evident at term gestation (E150). Brain and cerebellum weights of affected fetuses from unaffected mothers were not decreased. T4 and FT4 levels in affected fetuses were dependent on the maternal phenotype, as was the degree of enlargement of the goiter at E150. Newborn unaffected lambs from affected mothers had plasma T4 levels within the normal range. The present data show that in late gestation, fetal goats...

The effect of cervical X-irradiation on activity index of thyrocytes and plasma TSH: A pre-clinical model for radiation-induced thyroid damage

Because radiotherapy in the head and neck region is necessary in the treatment of childhood cancer, possibilities to prevent damage to the thyroid gland must be explored. We developed a model in which radiation-induced effects can be investigated in a way that these effects can be quantified, using thyroid dysmorphology and plasma TSH. Thirty-five Wistar rats, 5 weeks old, were X-irradiated on the cervical region, with a single dose varying from 0 to 20 Gy. After 6 weeks, TSH, T4 and T3 were determined, and thyroid glands were processed for histological examination by two independent pathologists. A histological classification scale was developed, using follicular size, colloid density and cell height of thyrocytes to measure hyperplasia and hypertrophy. By the sum of these scores, a cell-activity index was calculated, which was related to plasma TSH concentration. Numbers of PAS-positive droplets and epithelial desquamation were also counted. Inter-observer reliability was assessed. Good to very good reliability was found for scores of follicular size, colloid density and cell height. Significant increase of cell-activity index was found after 10, 15 and 20 Gy. The plasma TSH concentration was positively correlated to the cell-activity index, increasing with radiation-doses up to 15 Gy. The number of desquamated cells was significantly increased after radiation doses >10 Gy, with moderate reliability. In conclusion, this model using cell-activity index of thyrocytes together with plasma thyrotropin concentrations and desquamation of cells can be used for interpretation and future (pre-clinical) studies of prevention of radiation-induced thyroid damage.