Development and evaluation of a direct immunofluorimetric assay for urinary growth hormone.

We describe a two-site immunofluorimetric assay for urinary growth hormone that is resistant to interference from a wide range of urinary constituents and therefore eliminates the need for sample pre-treatment. A microtitre plate format is used with specific orientation of capture antibody on a polystyrene surface carrying a hydrazide group. Europium-labelled F(ab)2 is the fluorophore and time-resolved fluorimetry with co-fluorescence enhancement the signal detection system. Inter-assay precision was 11.3% at 5.2 ng/L and 10.3% at 44.3 ng/L, minimum detection limit (22% coefficient of variation, CV) was < 1.0 ng/L, working range (< 10% CV) was 0-100 ng/L and quantitative recovery and good parallelism were demonstrated. This convenient and sensitive assay is suitable for the routine measurement of human growth hormone (hGH) in urine.

Antiepileptic drug therapy and sexual function in men with epilepsy.

PURPOSE: To study the effects of antiepileptic drugs (AEDs) on sex hormone levels and sexual activity in a group of men attending a hospital-based epilepsy clinic. METHODS: One hundred eighteen men being treated with AED therapy, 32 with epilepsy but not receiving AEDs, and 34 controls were recruited. All subjects were aged 18-65 years. Blood (20 ml) was removed for hormone assays, after which each subject completed a validated questionnaire [Sexuality Experience Scores (Frenken and Vennix, 1981)] aimed at exploring the individuals' sexual activity and attitudes to sexual morality. RESULTS: Men taking carbamazepine (CBZ) only had significantly higher mean sex hormone-binding globulin (SHBG) levels than the control group. The CBZ group also had a significantly lower mean DHEAS concentration than the control, untreated, and sodium valproate (VPA) monotherapy groups. The phenytoin monotherapy group (PHT) had a significantly higher mean SHBG than both the control and untreated groups, and had a significantly higher mean total testosterone (TT) value than the control untreated, CBZ, and VPA groups, and a significantly lower mean DHEAS than the controls, untreated, and VPA groups. Men receiving more than one AED had significantly higher mean SHBG concentrations compared with control, untreated, and VPA groups. In addition, the polytherapy group's mean TT was significantly higher than the control and VPA groups, although its mean DHEAS concentration was lower than the control, untreated, and VPA groups. There were no significant differences between the study groups in mean FT, Budrostenedione (AND), or estradiol levels. But the CBZ, PHT, and polytherapy groups had significantly lower mean free and rogen index (FAI) than the controls. The CBZ group had a lower mean FAI than the VPA group. The polytherapy group had a lower FAI than the untreated group. Sexuality Experience Scores (SES) showed that those men receiving AEDs embraced a stricter sexual morality than the controls and untreated, and expressed greater satisfaction with their marriages than the control and untreated groups. CONCLUSIONS: Seizure type did not affect SES scores. Multiple regression showed men who had received further education were less accepting of strict sexual morality.

Alteration of the circadian rhythm of intact parathyroid hormone and serum phosphate in women with established postmenopausal osteoporosis.

Several studies have established that the circulating concentration of intact parathyroid hormone, PTH (1-84), over 24 h follows a circadian rhythm. The importance of this circadian rhythm is not known although some authors have detected alterations in the rhythm in metabolic bone disease and following dietary manipulation. We have studied the circadian rhythm of PTH (1-84) in 8 premenopausal women, 8 postmenopausal women with established osteoporosis and 8 postmenopausal women with no evidence of osteoporosis. Blood samples were obtained at 30-min intervals over a 24-h period and significant differences were found in the profiles of PTH (1-84) and serum phosphate in the three groups studied. Premenopausal women possessed a nocturnal/early morning increase in PTH (1-84) and phosphate (between 2200 and 0700 hours), as did postmenopausal women without osteoporosis. In postmenopausal women with osteoporosis the nocturnal increase in PTH (1-84) and serum phosphate was absent and PTH (1-84) decreased during the period 2200-0700 hours. A shift in acrophase is observed between premenopausal and postmenopausal women without osteoporosis. No acrophase was found in postmenopausal women with osteoporosis for either PTH (1-84) or serum phosphate. No circadian rhythm, acrophase or significant amplitude was observed in serum adjusted calcium or ionized calcium in any group studied. Alterations in the circadian rhythms for PTH (1-84) and serum phosphate occur in patients with postmenopausal osteoporosis that suggest that normal dynamics of PTH (1-84) secretion may play a role in both calcium and phosphate metabolism and the bone remodelling process. Whether these changes are causative or a response to the pathology will require further investigation.

Progesterone resistance in women who have had breast cancer.

OBJECTIVE: To investigate whether certain physiological responses to luteal progesterone are normal in women previously treated for breast cancer. DESIGN: Salivary progesterone concentrations, basal body temperatures, and breast blood flow changes (surface temperature method) were all recorded daily for one natural menstrual cycle. SETTING: Participants in the study made saliva collections and temperature measurements at home under semi-standardised conditions with supervisory visits by a project nurse. PARTICIPANTS: Twenty-five controls were compared with 30 women with previous breast cancer; all but three participants were parous and the average ages were 39 years (range 28-48) and 40 years (range 29-46), respectively. On average the women with previous breast cancer had had surgery 2.4 years previously; the operation was usually mastectomy, leaving the contralateral breast for study. RESULTS: Follicular phase (day 1-14) oral temperature averages were statistically indistinguishable between women in the control group and those with previous breast cancer. Luteal progesterone profiles were considered in the normal range for the controls and patients. However, the women with previous breast cancer, on average, exhibited a significantly smaller rise in the luteal phase basal body temperature. Follicular phase breast surface temperature was significantly higher in the breast cancer group (+0.30 degree C). This group showed a highly significant reduction of the luteal heat cycle in their breasts. CONCLUSIONS: Two progesterone-mediated physiological mechanisms have been found to be significantly less responsive in women with previous breast cancer than controls. The literature has been reviewed. Progesterone resistance could be a clinical entity and could be important in carcinogenesis.

Abnormal epinephrine release in young adults with high personal and high parental blood pressures.

BACKGROUND: Increased activity of the sympathetic nervous system has been proposed as a cause of high blood pressure (BP) and may be related to diet and body weight. To determine the role of these factors in predisposition to high BP, we studied 100 young adults with high or low BP from families in which both parents had either high or low BP. METHODS AND RESULTS: Plasma catecholamine, glucose, and insulin levels were measured before and after an oral glucose load. There was a significant correlation between fasting plasma norepinephrine and mean arterial pressure (P=.001). Subjects with high BP, irrespective of parental BP, were heavier (P=.003) and fatter (P=.002) and had a greater rise in plasma insulin (P=.003) following glucose than those with low BP. Offspring with high BP whose parents also had high BP showed an unexpected rise in plasma epinephrine (P=.004) following glucose. This adrenal medullary response was not the result of high parental or high personal BP alone as it was not seen in offspring with low BP whose parents had high BP or in offspring with high BP whose parents had low BP. CONCLUSIONS: Irrespective of family history, high BP is associated with increased body weight and hyperinsulinemia and reflects personal environment and behavior. However, abnormal epinephrine release is characteristic of the combination of genetic, environmental, and behavioral factors that is associated with high personal BP and a familial predisposition to high BP.

Sexual function in women with epilepsy.

PURPOSE: To examine certain aspects of sexual behaviour and attitudes in a group of women with epilepsy using a validated questionnaire (Sexuality Experience Scales), and effects of antiepileptic drugs (AEDs) on sex hormone binding globulin (SHBG), total testosterone (TT), and free testosterone (FT). METHODS: One hundred ninety-five women all attending a hospital-based epilepsy clinic were recruited. One hundred fifty-nine received AED therapy and 36 women did not. A control group of 48 women was also recruited. All women completed the first two Sexuality Experience Scales (SES 1 and SES 2), and those women in stable heterosexual relationships completed SES 3 and SES 4. At the same time blood was taken for TT and SHBG levels. FT was calculated using the method of Nanjee and Wheeler (22). RESULTS: Women receiving AED therapy achieved significantly higher SES 1 and SES 2 scores than the rest of the women, suggesting that they adhered to a stricter sexual morality and were less open to psychosexual stimulation than the rest of the women treated. Women in the treated group were less likely to have received further education than the controls, a factor that influences SES 1 and SES 2 scores. There were no significant differences between the three groups on SES 3 or SES 4, with the exception of the orgasmic adequacy (OE) scale of SES 3 where those women in the treated group attained a higher mean score than the controls, suggesting that they found orgasm less satisfying. Women receiving AEDs had significantly higher SHBG and TT than controls and untreated patients. There were no differences in FT between the groups. Those women receiving AEDs also exhibited the expected midcycle rise in FT. There was no significant correlation between FT and the desired frequency of intercourse or enjoyment of it for the three groups. CONCLUSIONS: Women in the treated group appeared to be more "moral" and less open to sexual approach, but those who had regular partners appeared to desire and enjoy intercourse as much as the control and untreated groups. This could be explained by differences in demographic characteristics of the study groups or be indicative of a bimodal distribution of sexual interest in these women. As FT was within physiological norms for each study group this cannot be cited as a cause for hyposexuality.

A comparison of the effects of alfacalcidol treatment and vitamin D2 supplementation on calcium absorption in elderly women with vertebral fractures.

Although vitamin D supplementation in the frail elderly improves calcium absorption, suppresses parathyroid hormone, decreases bone loss and reduces the risk of fractures, such treatment may be ineffective in patients with vertebral osteoporosis, because of impaired vitamin D metabolism or resistance to the action of vitamin D metabolites on the bowel. We have therefore performed a randomized, single masked study comparing the effects of alfacalcidol treatment (0.25 micrograms twice daily) and vitamin D2 supplementation (500-1000 units daily) on calcium absorption and bone turnover in 46 elderly women (median age 69 years, range 64-79 years) with radiological evidence of vertebral fractures. Serum 25-hydroxyvitamin D increased significantly after 3 and 6 months of treatment with vitamin D2 (p < 0.001), but was unchanged in the group receiving alfacalcidol. Serum 1,25-dihydroxyvitamin D did not change significantly in either group over the study period. Fractional 45Ca absorption increased after 3 months of treatment with alfacalcidol (p < 0.05), but was unchanged with vitamin D2. There was also a reduction in plasma intact parathyroid hormone and serum alkaline phosphatase after 6 months of treatment with alfacalcidol (p < 0.05) which was not seen in the group receiving vitamin D2. Our study shows that vitamin D2 supplementation is ineffective in stimulating calcium absorption in elderly women with vertebral osteoporosis. By increasing calcium absorption in such patients, alfacalcidol may prove more effective than vitamin D in the management of vertebral osteoporosis.

Successful suicide by insulin injection in a non-diabetic.

We report the death of a young female non-diabetic after self-administration of insulin which led to the trial and conviction of her partner, who was himself an insulin-dependent diabetic. This brings out particular lessons for the pathologist in view of the rarity of such deaths, emphasizes the importance of careful laboratory analysis and illustrates a number of interesting legal points.

Insulin-like growth factor binding protein 1 response to acute insulin induced hypoglycaemia in type 1 diabetes.

OBJECTIVES: Insulin is believed to be the prime regulator of insulin-like growth factor binding protein 1 (IGFBP-1) secretion, and in normal subjects acute insulin induced hypoglycaemia exerts a rapid effect on concentrations of IGFBP-1, and may also influence insulin-like growth factor I (IGF-I) concentrations. The rise in IGFBP-1 concentrations in normal subjects following hypoglycaemia has been suggested to be due to suppression of endogenous insulin secretion. We have examined this further by studying diabetics with no endogenous insulin secretion. DESIGN: We have compared the IGFBP-1 response to acute insulin induced hypoglycaemia in normal subjects and patients with Type 1 (insulin dependent) diabetes mellitus. METHODS: Insulin tolerance tests were performed using a bolus of insulin (0.15 U/kg), in six control subjects and six patients with Type 1 diabetes. MEASUREMENTS: Serum levels of IGFBP-1, insulin, glucose, and IGF-I were measured at regular intervals during the insulin tolerance test. RESULTS: Blood glucose fell to a nadir which coincided with the onset of the acute autonomic reaction 'R' in both groups. The basal concentration of IGF-I was significantly lower in the diabetic group at 0.4 +/- 0.1 kU/l, compared to 0.9 +/- 0.1 kU/l in the control group, but there was no significant change in IGF-I concentrations in response to hypoglycaemia in either group. Hypoglycaemia provoked a fall in IGFBP-1 in patients with Type 1 diabetes, from 38 +/- 9 micrograms/l basally to 17 +/- 3 micrograms/l at R + 120 minutes, with a return to basal values of 45 +/- 11 micrograms/l at R + 180 minutes. In the control subjects there was no fall in IGFBP-1, but a significant increase to 71 +/- 14 micrograms/l at R + 180 minutes. CONCLUSION: This difference in the IGFBP-1 response in the presence of a similar glucose response suggests that in Type 1 diabetes there may be different sensitivities to the actions of exogenous insulin on IGFBP-1 regulation.

The effect of recombinant insulin-like growth factor I on ketone body, lipid and apolipoprotein concentrations and its use to treat ketoacidosis in severe insulin resistance.

The effect of recombinant insulin-like growth factor I (rhIGF-I) on ketone body concentrations was studied in a patient with the Mendenhall syndrome, a rare insulin-resistant state. Treatment with intravenous rhlGF-I for an episode of ketoacidosis led to a clinical and biochemical improvement. One month later, the effect of 20 mg rhlGF-I infused daily for 4 days on ketone body concentrations was studied. From peak concentrations 24 h prior to the study to a nadir 72 h after the infusion commenced, acetoacetate fell from 4.17 mmol l-1 to 0.86 mmol l-1, beta-hydroxybutyrate from 9.91 mmol l-1 to 2.03 mmol l-1, and acetone from 2 mmol l-1 to 0.4 mmol l-1. Further studies of rhlGF-1 use caused a fall in concentrations of cholesterol, triglyceride, VLDL, LDL, and apolipoprotein B. Infusion of rhlGF-1 reduces ketone body concentrations and may be life-saving in the treatment of ketoacidosis developing in a patient with a severe insulin-resistant state.

Alteration of the circadian rhythm of intact parathyroid hormone following a 96-hour fast.

OBJECTIVE: PTH(1-84) secretion in normal male subjects follows a circadian rhythm. The control of this rhythm is multifactorial with both neuroendocrine and chemical influences. The aim of this study was to assess the effect of a 96-hour fast on the circadian rhythm of PTH(1-84), serum calcium, phosphate and nephrogenous cAMP (NcAMP), an index of PTH(1-84) bioactivity. DESIGN: Blood samples for estimation of all analytes were obtained over a 24-hour period at 30-minute intervals. Urine samples were obtained 4 hourly during the daytime and overnight. Each subject was studied on two occasions after being randomized to either (a) normal hospital diet or (b) a 96-hour fast with water freely available. SUBJECTS: Six healthy adult males aged between 28 and 40 years, mean 32 years. MEASUREMENTS: PTH(1-84) was measured by an in-house immunoradiometric assay. Serum calcium, phosphate, albumin, creatinine and urinary creatinine were measured by standard automated techniques. Calcium was adjusted for albumin. Plasma cAMP was estimated by a commercial method and urine cAMP by in-house radioimmunoassay and NcAMP obtained by calculation. Rhythm parameters were analysed by cosinor techniques. RESULTS: There were alterations in the circadian rhythms of serum phosphate, PTH(1-84) and NcAMP following a 96-hour fast. Fasting abolished the nocturnal rise in phosphate, PTH(1-84) and NcAMP but had little effect on the pattern of adjusted calcium over a 24-hour period. The mean concentrations of serum phosphate, adjusted calcium and NcAMP decreased significantly following the fast and mean PTH(1-84) increased during day time. CONCLUSIONS: Fasting for 96 hours significantly alters the circadian rhythm of PTH(1-84) secretion by lowering the mean calcium concentration and attenuating the circadian rhythm of serum phosphate.

Circulating intact parathyroid hormone levels in acute pancreatitis.

Serum levels of parathyroid hormone (PTH), calcium and albumin were measured daily for 5 days in 41 selected patients with moderate to severe acute pancreatitis. The PTH level was measured by means of a two-site immunoradiometric assay specific for the intact polypeptide. A rise in PTH level was observed more commonly in patients with a complicated or fatal outcome than in those with an uncomplicated course (14 of 16 versus six of 25 patients, P < 0.001). Although PTH levels were variable in the presence of hypocalcaemia, raised concentrations were found more frequently in patients with complications (seven of eight versus two of seven without complications, P = 0.035). This study confirms that an appropriate rise in PTH level occurs in response to the hypocalcaemic stimulus in patients with acute pancreatitis.

ACP Broadsheet No 141: September 1993. Role of endocrine biochemistry laboratories in the investigation of infertility.

The staff and services of the endocrine biochemistry laboratory are essential to the efficient investigation of infertility. Each centre should adopt a detailed strategy for the investigation of the infertile couple which specifies the hormone analyses required at each stage. Appropriate first-line hormone tests should be selected after a thorough clinical history and physical examination of both partners. Second-line hormone testing should be determined from the results of the initial investigation and should be restricted to requests that either confirm or clarify an endocrine basis to infertility or monitor the response to treatment. The clinical biochemist should advise on specimen timing and collection, have responsibility for guaranteeing time and valid hormone results, and be part of the team that audits the overall strategy and the outcome for individual patients.

Clinical and laboratory studies of a new immunoradiometric assay of parathyroid hormone-related protein.

Parathyroid hormone-related protein (PTHrP) was measured in plasma by a new immunoradiometric assay (IRMA) from Nichols Institute. The assay is specific for PTHrP and shows excellent parallelism when measuring keratinocyte fluid, samples with high PTHrP content, and PTHrP-supplemented plasma. A precision profile established the assay detection limit at 0.7 pmol/L. PTHrP was unstable in plasma, but the degradation rate was patient-specific. Because delay in separation resulted in loss of PTHrP immunoreactivity, samples were collected into tubes containing protease inhibitors (aprotinin, leupeptin, pepstatin, and EDTA) and separated within 30 min. Among normal subjects, 78% had PTHrP values greater than the detection limit; the reference range established was < 0.7-2.6 pmol/L. Of patients with hypercalcemia associated with malignancy, 46% had PTHrP > 2.6 pmol/L. PTHrP was increased in patients with breast (73%), genitourinary (64%), or lung (46%) malignancy but was rarely above normal in patients with hematological (29%) or gastrointestinal (33%) malignancy. PTHrP and nephrogenous cyclic adenosine monophosphate (NcAMP) were strongly correlated (r = 0.63, P < 0.01) in 37 patients with PTHrP values greater than the detection limit, but 8 patients had PTHrP and parathyroid hormone [PTH(1-84)] values below the limit of detection with inappropriate or increased NcAMP. Five of these eight patients had small cell carcinoma of lung. These patients may have secreted a factor that is not detected by the IRMAs of PTHrP or PTH used in this study but that produces hypercalcemia by means of cAMP-mediated mechanisms.

Pancreatic and pituitary hormonal responses to insulin-induced hypoglycaemia during muscarinic cholinergic blockade in man.

To investigate the role of muscarinic cholinergic mechanisms in mediating the pancreatic and pituitary hormonal responses to hypoglycaemia, six normal subjects were studied during acute insulin-induced hypoglycaemia under control conditions, and during blockade with intravenous atropine. During atropine blockade the response of pancreatic polypeptide was suppressed while the maximum response of plasma glucagon was significantly higher. The increment in plasma vasopressin was also increased significantly during cholinergic blockade. During blockade with atropine the responses of plasma prolactin was reduced, with a slight but significant reduction in the growth hormone response, and although a similar maximum response of plasma ACTH was achieved, this rise was delayed. These results implicate involvement of a cholinergic muscarinic inhibitory and stimulatory mechanisms in regulating the responses of pancreatic and pituitary hormones to hypoglycaemia.

Sleep shift dissociates the nocturnal peaks of parathyroid hormone (1-84), nephrogenous cyclic adenosine monophosphate, and prolactin in normal men.

The secretion of PTH(1-84) and PRL over a 24-h period in normal subjects has been shown to be highly correlated, with changes in PRL occurring approximately 2 h after those in PTH(1-84). It has been postulated that there may be neuroendocrine control common to PTH(1-84) and PRL. As the secretion of PRL is known to be strongly influenced by sleep we investigated the effect of a 7-h acute sleep shift on the nocturnal secretion of PTH(1-84), PRL, and nephrogenous cAMP, a marker of PTH(1-84) bioactivity. Six normal male subjects were studied on two occasions (study A sleep, 0100-0800 h; study B, 0800-1400 h) with samples withdrawn at 30-min intervals. Sleep shift produced the expected shift in PRL secretion to new time of sleep. The overall timing of the PTH(1-84) nocturnal peak (0200-0600 h) was not altered by sleep shift. However, the start of the rise in PTH(1-84) (0200-0300 h) was blunted (P less than 0.05), and the peak of nephrogenous cAMP, coincident with the nocturnal rise in PTH(1-84) in study A, was markedly attenuated (P less than 0.01). Thus whereas the results of this study argue against a direct neuroendocrine link between PTH(1-84) and PRL, it is postulated that sleep shift disrupts a high degree of temporal organization which, under normal conditions, may allow concerted metabolic effects between PTH(1-84) and other hormones over a 24-h period.