J. M. Burrin

Increased Hypothalamic Neuropeptide Y Concentrations in Diabetic Rat

Central and lateral hypothalamic concentrations of 10 regulatory peptides were measured by radioimmunoassay in streptozocin-induced diabetic (STZ-D) and matched control rats between 1 day and 14 wk after diabetes induction. After 2 wk, both central and lateral hypothalamic neuropeptide Y (NPY) concentrations in STZ-D rats were consistently higher than those found in control rats, with significant 30–50% increases at 4 wk in the central hypothalamus, and at 6 and 14 wk in both central and lateral hypothalamus. Immunocytochemical studies in 4- and 6-wk STZ-D animals showed the appearance of intensely NPY-positive swollen cell bodies in the supraoptic nucleus and a subjective increase in NPY staining of medial hypothalamic nerve fibers. Central hypothalamic concentrations of three other peptides were significantly greater in STZ-D animals than those in control animals at single points (neurotensin, 1 day; calcitonin gene-related peptide, 2 wk; neurokinin, 4 wk). Hypothalamic concentrations of the other six peptides examined (bombesin, galanin, neuromedin B, substance P, somatostatin, and vasoactive intestinal peptide) did not differ significantly between STZ-D and control groups at any time. However, galanin immunostaining in the supraoptic and magnocellular paraventricular nuclei was strikingly concentrated in a reduced number of distended cell bodies. Hypothalamic peptide changes in STZ-D could be related to metabolic disturbance, changes in energy and water balance, altered pituitary function, or other factors. Persistently elevated concentrations of NPY, a very potent central stimulant of eating and drinking, may mediate the hyperphagia and polydipsia characteristic of STZ-D.

EFFECTIVE LONG‐TERM TREATMENT OF ACROMEGALY WITH A LONG‐ACTING SOMATOSTATIN ANALOGUE (SMS 201–995)

Nine acromegalic patients, six previously untreated, were studied before and after 3–15 months of treatment with a long‐acting somatostatin analogue (SMS 201–995; 100 μg injected s.c. three times daily). During treatment, the mean (± SEM) 24‐h GH concentration fell from 82 ± 22 mIU/1 to 33 ± 7 mIU/1 (P < 0.001), and eight of the 9 patients showed a reduction of at least 50% in GH levels in the fasting state and/or during a glucose tolerance test. There was a significant 30% fall in serum concentrations of insulin‐like growth factor (IGF‐1) with SMS. All patients showed rapid clinical improvement, with diminished sweating and headaches, and reduction in skinfold thickness, hand volumes and finger size. Computer tomographic scanning of the pituitary in eight patients showed no change in the size of the pituitary tumour during treatment. The only side‐effects of SMS noted were transient abdominal discomfort and loose stools in two patients on initiating therapy. Although fasting plasma glucose concentration did not change during treatment (5.4 ± 0.3 vs 5.5 ± 0.3 mmol/l), mean 24‐h plasma glucose concentration was higher with SMS (6.6 ± 0.5 mmol/l vs 6.0 ± 0.4 mmol/l; P < 0.02). Mean 24‐h plasma insulin concentration fell from 87 ± 11 mIU/1 before treatment to 39 ± 6 mIU/1 during treatment (P < 0005). No change in other anterior pituitary hormones was observed. SMS appears to be a safe, rapidly effective, long‐term treatment for certain patients with acromegaly.

The responses of the catecholamines and β-endorphin to brief maximal exercise in man

SummaryThe responses to brief maximal exercise of 10 male subjects have been studied. During 30 s of exercise on a non-motorised treadmill, the mean power output (mean±SD) was 424.8±41.9 W, peak power 653.3±103.0 W and the distance covered was 167.3±9.7 m. In response to the exercise blood lactate concentrations increased from 0.60±0.26 to 13.46±1.71 mmol·l−1 (p<0.001) and blood glucose concentrations from 4.25±0.45 to 5.59±0.67 mmol·l−1 (p<0.001). The severe nature of the exercise is indicated by the fall in blood pH from 7.38±0.02 to 7.16±0.07 (p<0.001) and the estimated decrease in plasma volume of 11.5±3.4% (p<0.001). The plasma catecholamine concentrations increased from 2.2±0.6 to 13.4±6.4 nmol·l−1 (p<0.001) and 0.2±0.2 to 1.4±0.6 nmol·l−1 (p<0.001) for noradrenaline (NA) and adrenaline (AD) respectively. The plasma concentration of the opioidβ-endorphin increased in response to the exercise from <5.0 to 10.2±3.9 p mol·l−1. The post-exercise AD concentrations correlated with those for lactate as well as with changes in pH and the decrease in plasma volume. Post-exerciseβ-endorphin levels correlated with the peak speed attained during the sprint and the subjects peak power to weight ratio. These results suggest that the increases in plasma adrenaline are related to those factors that reflect the stress of the exercise and the contribution of anaerobic metabolism. In common with other situations that impose stress,β-endorphin concentrations are also increased in response to brief maximal exercise.

Direct assay for testosterone in saliva: relationship with a direct serum free testosterone assay.

A direct non-extraction radioimmunoassay for salivary testosterone is described using a modified commercial kit procedure that is in use for total serum testosterone (T). Serum free testosterone was also measured by direct radioimmunoassay. A significant correlation (r = 0.83, p less than 0.01, n = 194) was obtained between salivary and serum free testosterone in matched serum and saliva samples over a wide range of concentrations. Within- and between-batch precision for the salivary testosterone method was 11% and 18%, respectively at a concentration of 170 pmol/l. Recovery of added T was 89% +/- 15% (mean +/- 2 SD) dilution of high samples showed parallelism. Salivary testosterone measured by direct radioimmunoassay offers a simple cheaper alternative to serum free testosterone measurement with the additional advantages of a stress-free non-invasive sampling procedure.

The distribution of melanin-concentrating hormone-like immunoreactivity in the central nervous system of rat, guinea-pig, pig and man

Abstract The distribution of melanin-concentrating hormone-like immunoreactivity was investigated by radioimmunoassay in the CNS of rat, guinea-pig, pig and man. Highest concentrations of melaninconcentrating hormone-like immunoreactivity were found in the hypothalamus of all the species: rat204.4 ± 14.9; guinea-pig159.5 ± 23.3; pig10.9 ± 4.5 and man80.1 ± 19.1 pmol/g. Gel chromatographic analysis of hypothalamic extracts showed five immunoreactive peaks of melanin-concentrating hormone-like immunoreactivity in the rat and pig and six in the guinea-pig and man. High-performance liquid chromatography analysis of hypothalamic extracts showed five immunoreactive peaks in rat, guinea-pig, pig and four in man. However, these peaks appeared at different retention times from that of the single peak of salmon melanin-concentrating hormone. Examination of subcellular fractions of whole rat brain showed that most of the melanin-concentrating hormone-like immunoreactivity is found in the synaptosome fraction. Stimulation of melanin-concentrating hormone-like immunoreactivity release from rat hypothalamic slices revealed that potassium in the presence of calcium stimulated melanin-concentrating hormone-like immunoreactivity release. These findings suggest that mammalian melanin-concentrating hormone-like immunoreactivity has a different amino acid sequence from salmon melanin-concentrating hormone and may exist in multiple molecular forms. It is possible that melanin-concentrating hormone may play a role as a neurotransmitter or modulator in the mammalian CNS.

Evaluation of an enzyme immunoassay for plasma-free metanephrines in the diagnosis of catecholamine-secreting tumors

OBJECTIVE To define the test characteristics of an enzyme immunoassay (EIA) for plasma-free metanephrines (metanephrine and normetanephrine) in the diagnosis of pheochromocytoma and paraganglioma. DESIGN Prospective observational design from a single University Hospital. Twenty-four hour urine for catecholamines and plasma for free metanephrines were collected from patients with a clinical suspicion of pheochromocytoma or paraganglioma. Patient records were reviewed for clinical data, follow-up, imaging and laboratory results to establish or exclude the diagnosis of pheochromocytoma. PATIENTS AND METHODS Out of 178 consecutive patients, 10 had a paraganglioma and 12 had a pheochromocytoma: 156 were finally judged not to harbour active tumors and were therefore considered as controls. The main outcome measure was the diagnosis or exclusion of paraganglioma or pheochromocytoma and test characteristics of plasma-free metanephrines measured by EIA. RESULTS Urinary epinephrine had a sensitivity of 45.5% and norepinephrine a sensitivity of 75% (98.8% specificity) for the diagnosis of pheochromocytoma. Plasma-free metanephrine and normetanephrine both had a sensitivity of 66.7% and a specificity of 100%, but when combined (either positive) they demonstrated a 91.7% sensitivity with a preserved specificity of 100%. For the diagnosis of paraganglioma, urinary norepinephrine gave slightly better results than plasma-free metanephrines, but combined testing was of no additional value. CONCLUSIONS Plasma-free metanephrines measured by EIA have better diagnostic test characteristics than urinary catecholamines in the diagnosis of pheochromocytoma. The EIA offers a simple and effective measurement of plasma-free metanephrines.

Combined use of in situ hybridisation and immunocytochemistry for the investigation of prolactin gene expression in immature, pubertal, pregnant, lactating and ovariectomised rats

SummaryWe have investigated the use of in situ hybridisation together with immunocytochemistry for the study of endocrine cell function, using as an example the expression of prolactin messenger RNA (mRNA) in pituitaries of rats under various endocrinological conditions. In situ hybridisation using a 32P-labelled cRNA probe for rat prolactin was carried out on sections of 4% paraformaldehyde-fixed pituitaries from prepubertal, pubertal, pregnant, lactating and ovariectomised rats and adjacent sections were immunostained for prolactin. Northern gel analysis was performed on total RNA extracts of pregnant, lactating and control pituitaries. While in ovariectomised rat pituitaries both prolactin immunoreactivity and prolactin mRNA were decreased, no differences in prolactin immunostaining were seen between prepubertal, pubertal, pregnant or lactating rats and controls, even when the supra-optimal dilution technique was used. However, using in situ hybridisation, prolactin mRNA signal was increased in prepubertal rats, and with hybridisation and northern gel analysis the signal was reduced in pregnant rats and markedly increased in lactating rats. The combined use of in situ hybridisation and immunocytochemistry provides morphological information concerning endocrine gene expression and protein synthesis in the pituitary gland.

The matrix effects on kinetic rate constants of antibody–antigen interactions reflect solvent viscosity

This study describes the influence of different matrices on two model antibody-antigen interactions; that between beta2microglobulin and anti beta2microglobulin, and that of rabbit anti mouse Fc fragment (RAMFc) with mouse IgG. The matrices investigated were; phosphate-buffered saline pH 7.4 containing 0.05% Tween 20 detergent, horse serum, a 50:50 mixture of phosphate-buffered saline/Tween 20 and horse serum, and four glycerol solutions of differing concentrations. A recently developed optical biosensor, the IAsys, was used to monitor the interactions in real-time and provide precise determinations of k(ass), k(diss) and KA values. The results show that the rates of association and dissociation for the two different antibody:antigen models are significantly affected by the surrounding matrix. Glycerol of known viscosity was used as a matrix in both models to show that this effect is attributable to the viscosity as opposed to proteins present in the matrix. The viscosity of the matrix has also been shown to have an apparent influence upon the overall equilibrium/affinity constant for the interaction, with measurements of KA tending to increase with viscosity. The significant effects of matrix on kinetic rate constants for antibody-antigen interactions shown here have important implications in the use of immunoassays where non-equilibrium measurements are made in serum matrices.

The pituitary-thyroid axis in severe falciparum malaria: evidence for depressed thyrotroph and thyroid gland function

Abnormal thyroid function is strongly associated with mortality in severe non-thyroidal illness. We have assessed the pituitary-thyroid axis serially in 18 Thai adults with severe falciparum malaria and in 18 matched controls. The admission total serum thyroxine (T4) concentrations of the patients (median [range]: 64 nmol/litre [less than 30-91]) were significantly lower than those of controls (81 nmol/litre [61-133]; 2P less than 0.01), and remained depressed until after fever and parasite clearance. Two patients who died in hospital had admission serum T4 concentrations less than 35 nmol/litre. The admission basal serum thyrotropin (TSH) levels of the patients (0.9 mU/litre [less than 0.2-3.1]) were similar to those of controls (1.3 mU/litre [less than 0.2-3.7], 2P greater than 0.1) and remained normal throughout fever and parasitaemia. Thirty-minute TSH increments during a thyrotropin-releasing hormone test on admission were reduced in 13 patients with severe malaria (4.1 mU/litre [0.7-8.1]) relative to those in convalescence (7.1 mU/litre [1.7-14.4], n = 10, 2P less than 0.01) and controls (5.6 mU/litre [3.3-12.9], n = 9, 2P less than 0.05). These findings suggest that thyrotroph and thyroid gland function are depressed during acute, severe malaria. As these changes may be an adaptation to accelerated catabolism, the role of thyroid replacement in such patients is uncertain.

Hormonal and metabolic responses to cardiac surgery with sufentanil‐oxygen anaesthesia

The effects of sufentanil, 10 and 20 μg kg‐1 on the hormonal and metabolic responses to coronary artery surgery were compared in 20 patients. The most important finding was that the changes in circulating β‐ endorphin, ACTH, cortisol, GH, glucose, lactate and glycerol concentrations during and after cardiac surgery were similar with both doses of sufentanil. Although sufentanil prevented a significant increase in plasma β‐endorphin, ACIH and cortisol values until 6 h after cardiopulmonary bypass (CPB), a significant increase in GH secretion occurred with the onset of CPB. Plasma insulin concentrations declined significantly after 30 min CPB, but recovered after 60 min CPB with the restoration of normothermia. Blood glucose values did not change during surgery before CPB, but started to rise with the onset of CPB and continued to increase significantly in the postoperative period. Changes in blood lactate and plasma glycerol concentrations primarily reflected the load of CPB and the effects of heparin, respectively. The results show that increasing the dose of sufentanil up to 20 μg kg‐1 does not result in better suppression of the endocrine and metabolic changes associated with cardiac surgery.