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J. Furmaniak, J. Bradbury and B. Rees Smith

Abstract. The possibility that sera from patients with autoimmune thyroid diseases contain autoantibodies to thyroid membrane proteins distinct from microsomal antigen and the TSH receptor has been investigated using (a) solid phase assay system based on human thyroid membranes and 125I-labelled protein A and (b) immunoprecipitation of detergent solubilized 125I-labelled thyroid membranes followed by gel electrophoresis and autoradiography. In the solid phase assay binding to membranes showed a highly significant correlation with binding to microsomes (r = 0.82; P < 0.001; N = 82) indicating that the interaction between the serum and the membranes was due principally to microsomal antibody binding to microsomal antigen contaminating the membrane preparations. However, there were some discrepancies suggesting that an additional antigen-antibody system was involved. This possibility was then investigated using immunoprecipitation of 125I-labelled thyroid membranes. A labelled protein with mol wt 54 K was specifically immunoprecipitated (relative to normal pool serum) by 3 out of 4 sera from patients with Graves' disease who showed high binding to thyroid membranes. A further 4 sera from such patients with low membrane binding affinity failed to immunoprecipitate the 54 K protein. Sera from some patients with Hashimoto's disease and some patients with rheumatoid arthritis and one patient with Addison's disease also immunoprecipitated the 54 K protein from solubilized thyroid membranes. These studies suggested that antibodies interacting with the 54 K protein contributed to the discrepancies between thyroid membrane and microsome binding. However, the 54 K protein was also immunoprecipitated from detergent solubilized membranes prepared from human placenta, skeletal muscle and adrenal tissue. Immunoprecipitation studies with antisera to cytoskeleton proteins suggested that the 54 K band was the intermediate filament protein desmin. Consequently, thyroid specific antibody-antigen systems distinct from those involving microsomal antibody (or thyroglobulin antibody) could not be detected in thyroid membranes by immunoprecipitation.

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Q. T. Smith and Donna J. Allison

ABSTRACT

The wet weight, dry fat-free weight and collagen content of the uterus, the collagen, salt-soluble collagen and hexosamine contents of the skin and the collagen content of the femurs from weanling and young adult rats subjected to 1 to 21 daily injections of 2 μg 17β-oestradiol benzoate/100 g body weight were compared to similar data from control animals. Changes in dry fat-free weight of the uterus following oestradiol administration varied with animal age and did not coincide with changes in wet weight of the uterus. The dry fat-free weight of the uterus, as a per cent of wet weight, was significantly decreased in both age groups of rats following a single injecton of oestradiol only. Increases in dry fat-free weight of the uterus of the oestrogen-treated rats were primarily from substances other than collagen.

After 14 days of oestradiol injections, collagen per total skin was decreased in both weanling and young adult steroid-treated rats. The decrease in salt-soluble collagen extracted per gram of skin collagen at the later experimental periods was not great enough to warrant the conclusion that the quantity of salt-soluble collagen, relative to total skin collagen, was decreased. Hexosamine was not significantly decreased in either age of oestrogen-treated rats until the 21 day experimental period. The dry fat-free femur weight and collagen per femur of the oestrogen-treated rats were significantly changed (increased) only at the 14 day experimental period of the young adult rats.

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A. J. Sempéré, G. A. Bubenik and J. H. Smith

Abstract. The plasma levels of thermolabile (TLAP) and thermostable (TSAP) alkaline phosphatase were investigated in adult male white-tailed deer. Distinct seasonal variation of TLAP (with highly elevated levels in July) were observed, whereas TSAP exhibits low concentration with a small increase in October. No circadian rhythm was found for TLAP or TSAP. A close correlation (r = 0.98) between TLAP activity and antler weight was found. Administration of ACTH or dexamethasone were ineffective in influencing AP activity. On the other hand, variations of triiodothyronine (T3) levels in plasma induced by thyroxine (T4) injections correlated well with concentration of TLAP.

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J. S. Dirch Poulsen, Mogens Smith, Marja Deckert and Torsten Deckert

Abstract.

In order to avoid complications induced by long-term infusions of insulin into the portal vein, we examined the effect of intraperitoneal (ip) insulin infusion on arterial plasma insulin and glucose concentrations in 6 pigs, made diabetic by a constant intravenous (iv) infusion of glucose, epinephrine and propranolol.

Insulin was infused by an electromechanical programmable mini-pump (Pharmaject Micro Infusion System®, Pharmacia Electronics) as a booster injection of 46 mU highly purified porcine insulin Leo®/kg body weight, followed by 3 infusion periods of 30 min each with stepwise decreasing infusion rates of 1.6–0.8 and 0.2 mU/kg/min in a total volume of 192 μ1. Insulin was infused in a peripheral vein, a portal vein and into the peritoneal cavity.

A steep rise of arterial plasma insulin was demonstrated followed by a slow and identical decline in the peripheral and portal experiments, whereas only a small increase of plasma insulin was seen in the ip experiment, indicating insufficient absorption of insulin from the peritoneal cavity. The decrease of plasma glucose was identical in the peripheral and portal vein experiments, indicating that insulin infused in the portal vein does not seem to have a higher hypoglycaemic effect, than insulin infused in a peripheral vein.

Intraperitoneal insulin infusion seems not to be a practical substitute for iv insulin infusion.

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K Obuobie, J Smith, R John, JS Davies and JH Lazarus

OBJECTIVE: To assess central arterial stiffness in thyrotoxicosis using the technique of pulse wave analysis. DESIGN: Case control study designed to determine the effect of thyrotoxicosis on central arterial stiffness and at 6 months after radioiodine treatment. PATIENTS: Twenty (18 women and 2 men) thyrotoxic patients and 20 age- and sex-matched controls were studied at baseline. Thyrotoxic patients were re-studied at 6 months following treatment of thyrotoxicosis with 555 MBq (131)I with no additional therapy for the six-month period. MEASUREMENTS: Using the sphygmocor apparatus, peripheral pressure waveforms were recorded non-invasively from the radial artery and central pressure waveforms were generated from these. Indices of arterial stiffness, central augmentation index (AI), augmentation of central arterial pressure (AG) and central blood pressures were derived. AI corrected for heart rate (AIc) was calculated. RESULTS: Thyrotoxic patients recorded a significantly lower AI (means+/-s.e.m.) compared with controls (15.0+/-2.1 vs 28.0+/-2.1%; P<0.0005) even when corrected for differences in heart rate AIc (20.0+/-2.1 vs 28.0+/-2.1%; P<0.005) as well as AG (6.0+/-0.8 vs 10.0+/-1.1 mmHg; P<0.002) but higher pulse pressure (58.0+/-3.5 vs 47.0+/-2.0 mmHg; P<0.02). At 6 months following treatment, a significant rise in AIc (27.0+/-1.8 vs 20.0+/-2.1%; P<0.005) and AG (11.0+/-1.0 vs 6.0+/-0.8 mmHg; P<0.005) was noted. Lipid profiles were comparable between the groups. CONCLUSIONS: These data suggested that subjects with untreated thyrotoxicosis have a decreased augmentation of central arterial pressure or lowered central arterial stiffness that would not appear to contribute to any excess cardiovascular risk in that condition.

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E Hogervorst, J Williams, M Combrinck and A David Smith

OBJECTIVE: Oestrogens could be protective against the development of Alzheimer's disease (AD) but reports on oestrogen levels in AD have been conflicting. DESIGN AND METHODS: A meta-analysis using robust regression was carried out to assess whether the sensitivity of the assays of past studies had affected the reported level of total oestradiol. We had also measured total oestradiol in women with AD (n=66) and controls (n=62) not using hormone replacement therapy. We used two assays for total oestradiol to assess the difference between sensitive (radioimmunoassay with a specific rabbit antibody: 3 pmol/l) and relatively insensitive (immunoassay: 37 pmol/l) assays. RESULTS: Meta-analysis using robust regression indicated that insensitive assays gave higher levels of total oestradiol when many samples fall below the level of sensitivity of the method. Earlier reports of low levels of total oestradiol in AD might be explained by this phenomenon, since total oestradiol levels (using the sensitive assay) in our controls were one third of those reported in the earlier studies. Using the sensitive assay we found that women with AD had significantly (P<0.01) higher levels (26+/-13 pmol/l) of total oestradiol than controls (21+/-13 pmol/l). Using the insensitive assay, there was no significant difference in the levels of total oestradiol. CONCLUSIONS: The sensitivity of the assay determines the reported value of the oestradiol levels. Studies using a sensitive assay do not report significantly lower levels of total oestradiol in women with AD. This weighs against the hypothesis that low levels of total oestradiol are a risk factor for AD.

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P. P. A. Smyth, D. Neylan, N. M. McMullan, D. F. Smith and T.J. McKenna

Abstract. The rare occurrence of hyperthyroidism with an autonomously functioning nodule which following 131I therapy presented as toxic diffuse goitre (Graves' disease) is described in a 60 year old male. This progression was characterised by the presence of varying concentrations of IgG thyroid stimulators, thyroid stimulating immunoglobulins and thyroid growth stimulating immunoglobulins, as measured by cytochemical bioassay. It is postulated that the presence of the nodule and its associated hypersecretion of thyroid hormones may have protected the gland from the effects of IgG stimulators by bringing about inhibitory short-loop feedback on normal thyroid cells. It is further suggested that following therapeutic ablation of the nodule, normal thyroid cells became sensitive to the thyroid stimulators with the evolution of typical features of toxic diffuse goitre.

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Luis J. Rodriguez-Rigau, David B. Weiss, Keith D. Smith and Emil Steinberger

ABSTRACT

Androgen biosynthesis in the testis may be analyzed in some detail by means of techniques of in vitro incubation of small testicular biopsy specimens with suitable radiolabelled precursors. Sixty-six tissue specimens from 33 patients who underwent bilateral testicular biopsies because of infertility were incubated in vitro with [3H]pregnenolone in order to investigate the possibility of abnormalities in their steroid biosynthetic activity. As a normal control, testicular tissue obtained by testicular biopsy from a young normal volunteer was used. The distribution of metabolites in the incubates of testes from 8 infertile men differed greatly from the remaining 25 patients and the normal control. The major steroids formed from pregnenolone by the testes of those 8 men were 17-hydroxypregnenolone, dehydroepiandrosterone, 20α-dihydropregnenolone and 20α-dihydro-17-hydroxypregnenolone. Very small amounts of Δ4-3 oxo products (progesterone, 17-hydroxyprogesterone, androstenedione and testosterone) were formed suggesting a deficiency of 3β-hydroxy-steroid-dehydrogenase activity in the testes of these 8 men, possibly related to the derangement of their spermatogenic function.

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Vicki L Clifton, Phillip C Owens, Phillip J Robinson and Roger Smith

Clifton VL, Owens PC, Robinson PJ, Smith R. Identification and characterization of a corticotrophinreleasing hormone receptor in human placenta. Eur J Endocrinol 1995;133:591–7. ISSN 0804–4643

Corticotrophin-releasing hormone (CRH) causes vasodilatation in the human fetal–placental circulation and has paracrine actions in placental tissue, suggesting that CRH receptors may be present in the human placenta. We have now identified and characterized placental CRH binding sites and compared them to those described previously in human myometrium and rat pituitary. Radiolabelled ovine CRH binding to placental membranes was pH-, time-, temperature- and divalent cation-dependent and was reversible in the presence of 1 μmol/l unlabelled ovine CRH. Scatchard analysis of placentae delivered vaginally or by elective caesarean section revealed dissociation constants (Kd) of 214.5 ± 84 pmol/l (N = 8) and 45.4 ± 23.9 pmol/l (N = 9), respectively. The Kd for caesarean placental binding sites was similar to that of human myometrium (59.6 pmol/l, N = 3) and rat pituitary (82.5 pmol/l, N = 3) receptors. However, in vaginally delivered placentae the CRH binding sites had a much lower affinity (p < 0.05). The receptor densities (Bmax) of vaginally delivered and caesarean-delivered placentae were 28.6 ± 9.6 and 6.1 ± 2.8 fmol/mg, respectively (p < 0.05). Chemical cross-linking studies using disuccinimidyl suberate indicated that the molecular weight of the CRH receptor in the placenta and rat pituitary is 75 kD. We conclude that there is a high-affinity population of CRH binding sites in the human placenta that are physicochemically similar to pituitary and myometrial CRH receptors. The CRH receptor properties in the placenta change in response to labour, when CRH levels in maternal blood are highest, suggesting that placental CRH may regulate its receptor.

R Smith, Endocrinology Unit, John Hunter Hospital, Locked Bag 1, Hunter Regional Mail Centre, Newcastle, NSW 2310, Australia

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Cynthia G. Goodyer, John S. Torday, Barry T. Smith and C. J. P. Giroud

ABSTRACT

Bovine adrenocortical cells dispersed by trypsin digestion of fasciculata-reticularis minces were maintained in monolayer culture for up to 6 weeks.

During the first week cells grown in medium containing ACTH (1 mU/ml) secreted steroids at a rate 10 to 20-fold greater than control cultures, cortisol accounting for 80–90% of the corticotrophic response. Using tracer amounts of [3H]progesterone and [3H]pregnenolone, the major products were cortisol, corticosterone, 11-deoxycortisol and 11-deoxycorticosterone in decreasing order of magnitude.

After 10 to 15 days in culture steroidogenesis was no longer enhanced by ACTH. This was concomitant with an apparent loss of 11β-hydroxylase activity which was mainly manifested by a sharp increase in the formation of 11-deoxycortisol.

Short-term incubations of these cells during the first week in culture provided evidence that steroidogenesis was related to ACTH concentrations (from 0.1 to 100 μU/ml) and stimulated by dibutyryl cyclic AMP, the corticotrophic responses being further enhanced by theophylline (0.5 to 50 μmoles/5 ml). Exposure of the cells to ACTH (50 μU/ml) resulted in a rapid increase in intracellular cyclic AMP concentrations concomitant with a progressive increase in the corticosteroids released into the medium.

The data are consistent with the conclusion that during the first week in culture these cells provide a valuable model for the study of factors regulating steroidogenesis in the zona fasciculata-reticularis.