Dieter Schönberg

Transient Secondary Hypothyroidism in Children after Cardiac Surgery

Thyroid hormone status was assessed in 132 children with congenital heart defects undergoing cardiac surgery (median age 3.1 y; range 2 d to 16.2 y). Plasma TSH, thyroxine (T4), free thyroxine (fT4), triiodothyronine (T3), reverse triiodothyronine (rT3), thyroglobulin (Tg), and urinary iodine excretion were measured before and every other day after cardiac surgery (d 1-21). After surgery we observed strikingly low plasma concentrations of TSH (0.4 mU/L; 0.2-1.3), T3(0.6 nmol/L; 0.3-1.2), T4 (48.9 nmol/L; 12.9-82.4), fT4 (12.9 pmol/L; 5.1-19.3), and Tg (9.4 μg/L; 1.5-20.6), whereas rT3 plasma concentrations increased (0.13 pmol/L; 0.05-0.3; n = 40). The maximal postoperative changes of TSH and rT3 preceded changes of T3, T4, fT4, and Tg. Postoperative urinary iodine excretion increased significantly (n = 109). Thyroid hormone plasma concentrations were lowest after cardiopulmonary bypass operations and in patients treated with dopamine. In patients with postoperative T3 plasma concentrations less than 0.6 nmol/L (n = 52) the period of mechanical ventilation and intensive care treatment was significantly prolonged. Furthermore, the cumulative doses of inotropic and vasoactive catecholamines and furosemide were significantly higher in this patient group. Our results demonstrate transient secondary hypothyroidism in children after cardiac surgery that may contribute to postoperative cardiac and respiratory dysfunction and may delay recovery. Possible benefits of thyroid hormone replacement therapy need to be thoroughly examined.

Influence of antibodies against IGF-I, insulin or their receptors on proliferation of human acute lymphoblastic leukemia cell lines

To evaluate the potential role of IGF-I and insulin as growth-promoting factors in malignant lymphocyte development, we examined established T-acute lymphoblastic leukemic (ALL) cell lines with increasing stage of differentiation, HSB2, HUT78, CEM, MOLT3, Jurkat, JM-P, JM-886, and four established preB- and B-ALL cell lines REH, SKW6 CESS, BJAB for production of IGF-I and growth in the presence of antibodies, directed against IGF-I or insulin or their receptors. Basal DNA synthesis of the early differentiated T-cell lines HSB2 and HUT78, as well as the B-cell line REH, could be inhibited in a dose-dependent manner by both monoclonal antibodies against IGF-I (ASC41) and antibodies against the IGF-I receptor (alpha-IR3), suggesting that IGF-I acts as an auto- or paracrine growth factor for these cells via the IGF-I receptor. From these cells HUT78 and REH secreted IGF-I into cell culture medium. DNA synthesis of the further differentiated T-cell lines CEM and MOLT3 was inhibited by alpha-IR3 and antibodies directed against the insulin receptor (RPN.538) and against insulin (RPN.1661). These results suggest that insulin via the IGF-I receptor or insulin receptor can function as an autocrine or paracrine growth factor in these T-ALLs. Proliferation of the most differentiated T-ALL Jurkat and JMP was inhibited only by alpha-IR3 and 2C2, an antibody directed against the IGF-II receptor, suggesting that IGF-I or IGF-II acting via the IGF-I receptor or IGF-II receptor may be involved in proliferation of these cell lines. Inhibition of the DNA synthesis by RPN.538 and RPN.1661 indicate a more important role for insulin in growth of leukemias of the B-ALL cell lines SKW6 and CESS.

Characterisation of insulin-like growth factor I receptors of human acute lymphoblastic leukaemia (ALL) cell lines and primary ALL cells

The expression of insulin-like growth factor I (IGF-I) receptors (IGR-IR) on human B-lineage and T-lineage acute lymphoblastic leukaemias (ALL) representing different maturational stages has been studied. Immature (stage I) and mature (stage II) T ALL as well as pre-B ALL cell lines expressed high numbers of IGF-IR with high affinity for IGF-I. In contrast, on T ALL, stage II and B ALL only low specific binding of 125I-IGF-I was detected. No binding of 125I-IGF-I to Burkitt lymphoma cells was found. Primary human T, pre-B and cALL cells also expressed IGF-IR with Kd for IGF-I and IGF-IR number per cell in the same range as the investigated cell lines. Crosslinking of 125I-IGF-I to T and pre-B ALL cells revealed IGF-IR alpha-subunits of 135 and 116 kD for HSB2. Gene expression of IGF-IR could be detected in all T ALL cell lines but was undetectable in SKW6, a B ALL cell line.

Short-term, high-dose testosterone treatment fails to reduce adult height in boys with constitutional tall stature.

Abstract Height predictions based on three different methods (Bayley-Pinneau [BP], Tanner-Whitehouse Mark II [TW II], Roche-Wainer-Thissen [RWT]) were compared to adult heights in 19 males with constitutional tall stature previously treated with high-dose testosterone oenanthate for 6 months (group A) and 25 untreated tall males (group B). Their chronological ages (CA) at the initial evaluation of tall stature ranged from 12.1 to 16.6 years in group A and from 10.4 to 15.7 years in group B; at the time of assessment of adult height ages ranged from 18.0 to 26.5 years and from 18.4 to 25.1 years, respectively. Height measurements and predicted adult heights were expressed as height standard deviation scores (height SDS) for chronological age using the tables of Reinken and van Oost [14]. Height SDS in group A were 2.8 (range = 1.8–5.4) before testosterone treatment, 3.0 (range = 2.0–4.8) thereafter and finally 3.0 (range = 2.1–4.2) (P=NS) and in group B 2.7 (range = 0.5–4.3) and 2.4 (range = 1.3–3.5) (P=NS). A significant difference between adult height SDS and predicted height SDS according to BP was detected both in group A (3.0; range = 2.1–4.2 vs 3.6; range = 2.4–5.0; P≤0.004) and group B (2.4; range = 1.3–3.5 vs 3.0; range = 2.0–4.9; P≤0.0002), whereas no significant difference between adult height SDS and predicted height SDS according to TW II and RWT was found in either group. These data indicate that BP height predictions overestimated adult height in our patient group of treated and untreated males with constitutional tall stature. In contrast, the TW II and RWT methods were more accurate in predicting adult height in these patients, but also failed to demonstrate that testosterone therapy in boys with constitutional tall stature can be limited to a 6-month period in order to reduce adult height. Conclusion The widely used height prediction method of BP is inaccurate in boys with constitutional tall stature. High dose testosterone treatment fails to reduce adult height in these individuals when discontinued before complete closure of the epiphyses.

Demonstration of type I insulin-like growth factor receptors on human platelets.

Human platelets, freshly isolated from healthy human adults, express receptors for insulin-like growth factor I. The IC50 for displacement of 125I-IGF-I binding by unlabeled IGF-I was 0.2 nM, by IGF-II 32 nM by insulin 160 nM. Scatchard analysis of IGF-I binding demonstrates dissociation constants of 0.14 +/- 0.08 nM for high affinity binding site and 54 +/- 18 nM for low affinity binding site. The presence of the alpha-subunit of type I IGF receptor, as high affinity binding site, was verified by affinity crosslinking of 125I-IGF-I to platelet surface membranes. Under reducing con-conditions a Mr = 135,000 band was preferentially labeled. The complete type I IGF receptor complex, which revealed under nonreducing conditions, has an approximately molecular mass of Mr greater than 400,000. The immunoprecipitation of the 125I-IGF-I cross-linked type I receptor with alpha IR-3 confirmed the results achieved by affinity crosslinking.

Preparative separation of human B and T lymphocytes by free flow electrophoresis.

An electrophoretic method for the quantitative separation of human B and T lymphocytes in a carrier-free system is presented. The method is based on the fact that B and T lymphocytes show marked overlap in their size and density characteristics, but differ sufficiently in surface charge to be separable by electrophoresis. The technique is performed in phosphate-buffered saline and appears to be especially suitable for the enrichment of nonstimulated, functionally intact lymphocytes which can be directly used for further immunological or biochemical studies.

49 INSULIN-LIKE GROWTH FACTOR(IGF)AND CARRIER PROTEIN SERUM LEVELS IN TALL GIRLS ON HIGH-DOSE ESTROGENS

To further elucidate the behavior of IGF under highdose estrogens we have determined total IGF (tIGF) by a competitive protein binding assay, IGF-I by RIA and IGF carrier protein levels by a modification of the protein binding assay in 17 excessively tall girls (mean age ± SD:13.9 ± 1.3 yrs, mean bone age ± SD:12.4 ± 0.6 yrs) before and 12 months after initiation of estrogen treatment (ethinylestradiol (EE) 300 μg per day). Before therapy the mean ± SD serum level of tIGF was 427 ± 43.4 μU/ml,of IGF-I 182.9 ±36.4 ng/ml.tIGF decreased to 370.7 ± 72 μU/ml(86% of level before) ,IGF-I to 128.3 ± 19.3 ng/ml(70% of level before).Carrier protein concentrations did not change significantly (430.3 ±58.4 ngeq/ml vs. 415.9 ± 77.5 ngeq/ml,n.s.) There was a significant positive correlation between Δ IGF-I and growth rate(r=0.55,p < 0.05) and a significant negative correlation between Δ IGF-I and growth rate(r=0.66, p < 0.001) on EE.- Our results are in keeping with the assumption that growth inhibition observed with highdose estrogens is at least in part due to a specific effect on IGF production.While the net carrier protein content is unchanged, the possibility has to be tested that IGF metabolism may be increased by a change of the carrier protein composition.

SECONDARY HYPOTHYROIDISM IN PEDIATRIC CARDIAC SURGERY PATIENTS

Normal thyroid function is vital for growth and nervous system myelinization. The number of myocardial β-adrenergic receptors and the rate of synthesis and use of myocardial high-energy phosphates are thyroid hormone dependent. Abnormal thyroid function may occur after cardiac surgery; we therefore assessed perioperative thyroid hormone secretion in children. 82 patients (age range 2 days to 16 years) with congenital heart disease were studied before and after surgery (day 1, 3, 5, 7). Plasma TSH, T3, T4, fT4, TG and urinary iodine excretion were measured. Results are expressed as mean ± SEM. Statistical analysis was performed by ANOVA (preoperative vs. postoperative) and a general linear models procedure (SAS).There was also a significant fall of fT4 and TG. T3/T4 ratio remained unchanged. Plasma hormone values failed to reach preoperative values within 7 days. This effect was statistically independent of age, cardiopulmonary bypass and iodine contamination. TSH(R=0.43), T3(R=0.74), T4(R=0.56), fT4(R=0.42), TG(R=0.42)were significantly lower in dopamine treated patients compared to children not receiving dopamine infusion. We conclude, that secondary hypothyroidism was present in all patients. T3 replacement therapy has to be strongly considered in pediatric patients after cardiac surgery, especially when treated with dopamine. T3 may serve as a positive inotropic agent and reduce inotropic support (catecholamines). Whether this therapy will influence the postoperative intensive care course of these patients in general and will reduce recovery time from surgery is under investigation.

DIANGOSTIC OF IGF-1 INDUCTION IN GROWTH RETARDED CHILDREN: CHANGES OF IGF-1 RECEPTOR NUMBER AND BINDING AFFINITY AFTER 5 DAYS GH ADMINISTRATION

The acute response of IGF-1 to GH treatment (5 days, 0.1 E/kg per day) has been tested in seven children with growth retardation. All chlldrens have a normal GH-stimulation . With the change of the IGF-1 level after GH administration we tested the change of the IGF-1 receptor number and -affinity on the isolated lymphocytes of these patients. The IGF-1 level raised from 102 ± 57 ng/ml to 180 ± 94 ng/ml ≙ 186 + 65 %. In 5 patients there was no significant change in receptor affinity (from 0.47 ± 0.33 × 109 M−1 to 0.45 ± 0.31 × 109 M−1). Two subjects had a stimulation of receptor affinity from 0.28 ± 0.1 to 0.48 ± 0.15 · 109 M−1. The receptor number from 6 patients however decreased from 7282 ± 3309 to 4926 + 2538. This decrease was strongly correlated to the increase of the IGF-1 level (r= 0.94). One patient had an increase of receptor number from 2500 to 4980.Our results are corresponding with the suggestion of Rosenfeld et al (1982), that the downregulation of IGF-1 binding is accounted by a decrease in receptor number rather than change in binding affinity. It has to be discussed whether GH itself has an influence of increase of receptor affinity ( 2 patients) or receptor number ( 1 patient ).R.G. Rosenfeld et al (1982) Diabetes 31:375

A SIMPLIFIED METHOD FOR SMALL SCALE PURIFICATION OF INSULIN-LIKE GROWTH FACTOR I / SOMATOMEDIN C (IGF I/ SM C) FROM COHN FRACTION IV

Starting from Cohn fraction IV of human plasma we earlier described a multiple step procedure which resulted in ug amount of IGF I/SmC of relative high purity (200 μU/pg)+.In the meanwhile we have further modified this method: 20 grs. of acetone dried Cohn fraction IV are suspended in 1% NaCl/formic acid, pH 3.0. After precipitation of the IGF I containing proteins in the supernatant by addition of 5 M NaCl the precipitate is resuspended in 2 N acetic acid (AA) /ethanol (1:4). Denatured proteins are discarded by centrifugation, and after evaporation the supernatant is chromatographed on Sephadex G 50 in 1 N AA. IGF I containing fractions (approx. 12 μg IGF I, purity of approx. 90%) are purified by ImmobilineR isoelectric focusing (IEF) (pH 8-10). The IGF I band, focusing at pH 8.3 finally is purified using a HPLC reversed phase C-18 column in a 25-65% acetonitrile gradient cont. 0.05% TFA. IGF I elutes in a single peak at approx. 40% acetonitrile. Since the G 50 material contains no IGF II as demonstrated in preparative ImmobilineR IEF pH 4-10 gradient, even this material can be applied for antibody production. The final step material of highest purity is suitable for biological studies and in vitro investigation of IGF I receptor interactions on human lymphocytes and fibroblasts.Ref.: +P.Mayer, U.Heinrich, D.S.Schalch, D.Schönberg: Pediat. Res. 18, 107 (1984)