Analysis and application of an equilibrium model for in vitro bioassay systems with three components: Receptor, hormone and hormone-binding-protein

A simple theoretical framework is presented for bioassay studies using three component in vitro systems. An equilibrium model is used to derive equations useful for predicting changes in biological response after addition of hormone-binding-protein or as a consequence of increased hormone affinity. Sets of possible solutions for receptor occupancy and binding protein occupancy are found for typical values of receptor and binding protein affinity constants. Unique equilibrium solutions are dictated by the initial condition of total hormone concentration. According to the occupancy theory of drug action, increasing the affinity of a hormone for its receptor will result in a proportional increase in biological potency. However, the three component model predicts that the magnitude of increase in biological potency will be a small fraction of the proportional increase in affinity. With typical initial conditions a two-fold increase in hormone affinity for its receptor is predicted to result in only a 33% increase in biological response. Under the same conditions an Ii-fold increase in hormone affinity for receptor would be needed to produce a two-fold increase in biological potency. Some currently used bioassay systems may be unrecognized three component systems and gross errors in biopotency estimates will result if the effect of binding protein is not calculated. An algorithm derived from the three component model is used to predict changes in biological response after addition of binding protein to in vitro systems. The algorithm is tested by application to a published data set from an experimental study in an in vitro system (Lim et al., 1990, Endocrinology 127, 1287-1291). Predicted changes show good agreement (within 8%) with experimental observations. (C) 1998 Academic Press Limited.

Growth hormone binding protein correlates strongly with leptin and percentage body fat in GH-deficient adults, is increased by GH replacement but does not predict IGF-I response

GH-binding protein (GHBP) corresponds to the extracellular domain of the GH receptor (GHR) and has been shown to be closely related to body fat. This study aimed to examine the inter-relationship between GHBP, leptin and body fat, and to test the hypothesis that GHBP is modified by GH replacement in GH-deficient adults and predicts IGF-I response. Twenty adults, mean age 47 years (range 20-69) with proven GH deficiency were randomly allocated to either GH (up to 0.25 U/kg/week in daily doses) or placebo for 3 months before cross-over to the opposite treatment. Plasma GHBP and leptin were measured at baseline and 2, 4, 8 and 12 weeks after each treatment. Whole body composition was measured at baseline by dual-energy X-ray absorptiometry (DEXA). There was a strong correlation between baseline leptin and GHBP (r = 0.88, P < 0.0001) and between baseline GHBP and percentage body fat, (r = 0.83, P < 0.0001). Mean GHBP levels were higher on GH compared with placebo, 1.53 +/- 0.28 vs 1.41 +/- 0.25 nM, P = 0.049. There was no correlation between baseline IGF-I and GHBP (r = -0.049, P = 0.84), and GHBP did not predict IGF-I response to GH replacement. The close inter-relationship between GHBP, leptin and body fat suggests a possible role for GHBP in the regulation of body composition. GHBP is increased by GH replacement in GH-deficient adults, but does not predict biochemical response to GH replacement. (C) 1999 Churchill Livingstone.

Administration of growth hormone or IGF-I to pregnant rats on a reduced diet throughout pregnancy does not prevent fetal intrauterine growth retardation and elevated blood pressure in adult offspring

Increasing evidence from human epidemiological studies suggests that poor growth before birth is associated with postnatal growth retardation and the development of cardiovascular disease in adulthood. We have shown previously that nutritional deprivation in the pregnant rat leads to intrauterine growth retardation (IUGR), postnatal growth failure, changes in the endocrine parameters of the somatotrophic axis, and to increased blood pressure in later life. In the present study, we investigated whether administration of insulin-like growth factor-I (IGF-I) or bovine growth hormone (GH) during pregnancy could prevent IUGR and/or alter long-term outcome. Dams h-om day 1 of pregnancy throughout gestation received a diet of nd libitum available food or a restricted dietary intake of 30% of ad libitum fed dams. From day 10 of gestation, dams were treated for 10 days with three times daily subcutaneous injections of saline (100 mu l), IGF-I (2 mu g/g body weight) or GH (2 mu g/g body weight). Maternal weight gain was significantly increased (P

Placental growth hormone (GH), GH-binding protein, and insulin-like growth factor axis in normal, growth-retarded, and diabetic pregnancies: Correlations with fetal growth

We previously described significant changes in GH-binding protein (GHBP) in pathological human pregnancy. There was a substantial elevation of GHBP in cases of noninsulin-dependent diabetes mellitus and a reduction in insulin-dependent diabetes mellitus. GHBP has the potential to modulate the proportion of free placental GH (PGH) and hence the impact on the maternal GH/insulin-like growth factor I (IGF-I) axis, fetal growth, and maternal glycemic status. The present study was undertaken to investigate the relationship among glycemia, GHBP, and PGH during pregnancy and to assess the impact of GHBP on the concentration of free PGH. We have extended the analysis of specimens to include measurements of GHBP, PGH, IGF-I, IGF-II, IGF-binding protein-1 (IGFBP-1), IGFSP-2, and IGFBP-3 and have related these to maternal characteristics, fetal growth, and glycemia. The simultaneous measurement of GHBP and PGH has for the first time allowed calculation of the free component of PGH and correlation of the free component to indexes of fetal growth and other endocrine markers. PGH, free PGH, IGF-I, and IGF-II were substantially decreased in IUGR at 28-30 weeks gestation (K28) and 36-38 weeks gestation (K36). The mean concentration (+/-SEM) of total PGH increased significantly from K28 to K36 (30.0 +/- 2.2 to 50.7 +/- 6.2 ng/mL; n = 40), as did the concentration of free PGH (23.4 +/- 2.3 to 43.7 +/- 6.0 ng/mL; n = 38). The mean percentage of free PGH was significantly less in IUGR than in normal subjects (67% vs. 79%; P < 0.01). Macrosomia was associated with an increase in these parameters that did not reach statistical significance. Multiple regression analysis revealed that PGH/IGF-I and IGFBP-5 account for 40% of the variance in birth weight. IGFBP-3 showed a significant correlation with IGF-I, IGF-II, and free and total PGK at K28 and K36. Noninsulin-dependent diabetes mellitus patients had a lower mean percentage of free PGH (65%; P < 0.01), and insulin-dependent diabetics had a higher mean percentage of free PGH (87%; P < 0.01) than normal subjects. Mean postprandial glucose at K28 correlated positively with PGH and free PGH (consistent with the hyperglycemic action of GH). GHBP correlated negatively with both postprandial and fasting glucose. Although GHBP correlated negatively with PGH (r = -0.52; P

Effects of replacing meat with soyabean in the diet on sex hormone concentrations in healthy adult males

A randomised crossover dietary intervention study was performed to evaluate the effects of replacing meat protein in the diet with a soyabean product, tofu, on blood concentrations of testosterone, dihydrotestosterone, androstanediol glucuronide, oestradiol, sex hormone-binding globulin (SHBG), and the free androgen index (total testosterone concentration/SHBG concentration x 100; FAI). Forty-two healthy adult males aged 35-62 years were studied. Diets were isoenergetic, with either 150 g lean meat or 290 g tofu daily providing an equivalent amount of macronutrients, with only the source of protein differing between the two diets. Each diet lasted for 4 weeks, with a 2-week interval between interventions. Fasting blood samples were taken between 07.00 and 09.30 hours. Urinary excretion of genistein and daidzein was significantly higher after the tofu diet (P

Effects of an oral vasopressin receptor antagonist (OPC-31260) in a dog with syndrome of inappropriate secretion of antidiuretic hormone

Objective The syndrome of inappropriate secretion of antidiuretic hormone is a rare disorder in dogs characterised by hypo-osmolality and persistent arginine vasopressin production in the absence of hypovolaemia and/or hypotension. The study describes the efficacy and safety of the nonpeptide selective arginine vasopressin V-2 receptor antagonist OPC-31260 in a dog with the naturally occurring syndrome. Design The detailed case history of a dog with spontaneous syndrome of inappropriate secretion of antidiuretic hormone that received long-term therapy with oral OPC-31260 is presented. Effects of the first dose of OPC-31260 and of a dose administered after a continuous dosing period of 12 days are reported. Procedure Packed cell volume, plasma sodium, total protein, arginine vasopressin, renin activity, atrial natriuretic peptide, urine specific gravity, urine output, heart rate and body weight were monitored for 2 h before, and for 4 h after, the first dose of OPC-31260. The same parameters plus plasma osmolality and urine osmolality were monitored when an identical dose was administered after 12 days of therapy. Results Oral administration of OPC-31260 at 3 mg/kg body weight resulted in marked aquaresis with increased urine output and decline in urine specific gravity within 1 h. Corresponding increases in concentrations of plasma sodium, plasma osmolality and plasma renin activity were recorded over a 4 h period. Arginine vasopressin concentration remained inappropriately elevated throughout the study. Results were similar when the trial procedure was repeated after a stabilisation period of 12 days. Long-term therapy with OPC-31260 at a dose of 3 mg/kg body weight orally every 12 h resulted in good control of clinical signs with no deleterious effects detected during a 3-year follow-up period. Despite sustained clinical benefits observed in this case, plasma sodium did not normalise with continued administration of the drug. Conclusions Treatment of a dog with naturally occurring syndrome of inappropriate secretion of antidiuretic hormone with OPC-31260 at 3 mg/kg body weight orally every 12 h resulted in marked aquaresis and significant palliation of clinical signs with no discernible side-effects detected over a 3-year period. Thus, OPC-31260 appears to offer a feasible medical alternative to water restriction for treatment of dogs with syndrome of inappropriate secretion of antidiuretic hormone. Higher doses of OPC-31260 may be required to achieve and maintain normal plasma sodium in dogs with this syndrome.

Recombinant human growth hormone, but not insulin-like growth factor-I, enhances central fat loss in postmenopausal women undergoing a diet and exercise program

We examined the effect of recombinant human growth hormone (rhGH) and/or recombinant human insulin-like growth factor-I (rhIGF-I) on regional fat loss in postmenopausal women undergoing a weight loss regimen of diet plus exercise. Twenty-seven women aged 59-79 years, 20-40% above ideal body weight, completed a 12-week program consisting of resistance training 2 days/week and walking 3 days/week, while consuming a diet that was 500 kcal/day less than that required for weight maintenance, Participants were randomly assigned in a double-blind fashion to receive rhGH (0.025 mg/kg BW/day: n=7), rhIGF-I (0.015 mg/kg BW/day: n=7), rhGH + rhIGF-I (n = 6), or placebo (PL: n = 7). Regional and whole body fat mass were determined by dual X-ray absorptiometry. Body fat distribution was assessed by the ratios of trunk fat-to-limb fat (TrF/LimbF) and trunk fat-to-total fat (TrF/TotF), Limb and trunk fat decreased in all groups (p < 0.01). For both ratios of fat distribution, the rhGH treated group experienced an enhanced loss of truncal compared to peripheral fat (p less than or equal to 0.01), with no significant change for those administered rhIGF-I or FL. There was no association between change in fat distribution and indices of cardiovascular disease risk as determined by serum lipid/lipoprotein levels and maximal aerobic capacity. These results suggest that administration of rhGH facilitates a decrease in central compared to peripheral fat in older women undertaking a weight loss program that combines exercise and moderate caloric restriction, although no beneficial effects are conferred to lipid/lipoprotein profiles, Further, the effect of rhGH is not enhanced by combining rhCH with rhIGF-I administration. In addition, rhIGF-I does not augment the loss of trunk fat when administered alone.

Identification of cellular changes associated with increased production of human growth hormone in a recombinant Chinese hamster ovary cell line

A proteomics approach was used to identify the proteins potentially implicated in the cellular response concomitant with elevated production levels of human growth hormone in a recombinant Chinese hamster ovary (CHO) cell line following exposure to 0.5 mM butyrate and 80 muM zinc sulphate in the production media. This involved incorporation of two-dimensional (2-D) gel electrophoresis and protein identification by a combination of N-terminal sequencing, matrix-assisted laser desorption/ionisation-time of flight mass spectrometry, amino acid analysis and cross species database matching. From these identifications a CHO 2-D reference,map and annotated database have been established. Metabolic labelling and subsequent autoradiography showed the induction of a number of cellular proteins in response to the media additives butyrate and zinc sulphate. These were identified as GRP75, enolase and thioredoxin. The chaperone proteins GRP78, HSP90, GRP94 and HSP70 were not up-regulated under these conditions.

A randomized trial comparing hormone replacement therapy (HRT) and HRT plus calcitriol in the treatment of postmenopausal osteoporosis with vertebral fractures: Benefit of the combination on total body and hip density

We report a prospective, randomized, multi-center, open-label 2-year trial of 81 postmenopausal women aged 53-79 years with at least one minimal-trauma vertebral fracture (VF) and low (T-score below 2) lumbar bone mineral density (BMD). Group HRT received piperazine estrone sulfate (PES) 0.625 - 1.25 mg/d +/- medroxyprogesterone acetate (MPA) 2.5 - 5 mg/d,- group HRT/D received HRT plus calcitriol 0.25 mug bd. All with a baseline dietary calcium (Ca) of < I g/d received Ca carbonate 0.6 g nocte. Final data were on 66 - 70 patients. On HRT/D, significant (P < 0.001) BNID increases from baseline by DXA were at total body - head, trochanter, Ward's, total hip, inter-trochanter and femoral shaft (% group mean Delta 4.2, 6.1, 9.3. 3.7. 3.3 and 3.3%, respectively). On HRT, at these significant Deltas were restricted to the trochanter and sites. si Wards. Significant advantages of HRT/D over HRT were in BMD of total body (- head), total hip and trochanter (all P = 0.01). The differences in mean Delta at these sites were 1.3, 2.6 and 3.9%. At the following, both groups Improved significantly -lumbar spine (AP and lateral), forearm shaft and ultradistal tibia/fibula. The weightbearing, site - specific benefits of the combination associated with significant suppression of parathyroid hormone-suggest a beneficial effect on cortical bone. Suppression of bone turnover was significantly greater on HRT/D (serum osteocalcin P = 0.024 and urinary hydroxyproline/creatinine ratio P = 0.035). There was no significant difference in the number of patients who developed fresh VFs during the trial (HRT 8/36, 22%; HRT/D 4/34, 12% - intention to treat); likewise in the number who developed incident nonvertebral fractures. This Is the first study comparing the 2 treatments in a fracture population. The results indicate a significant benefit of calcitriol combined with HRT on total body BMD and on BNID at the hip, the major site of osteoporotic fracture.

Purification of recombinant human growth hormone from CHO cell culture supernatant by Gradiflow preparative electrophoresis technology

Purification of recombinant human growth hormone (rhGH) from Chinese hamster ovary (CHO) cell culture supernatant by Gradiflow large-scale electrophoresis is described. Production of rhGH in CHO cells is an alternative to production in Escherichia coli, with the advantage that rhGH is secreted into protein-free production media, facilitating a more simple purification and avoiding resolubilization of inclusion bodies and protein refolding. As an alternative to conventional chromatography, rhGH was purified in a one-step procedure using Gradiflow technology. Clarified culture supernatant containing rhGH was passed through a Gradiflow BF200 and separations were performed over 60 min using three different buffers of varying pH. Using a 50 mM Tris/Hepes buffer at pH 7.5 together with a 50 kDa separation membrane, rhGH was purified to approximately 98% purity with a yield of 90%. This study demonstrates the ability of Gradiflow preparative electrophoresis technology to purify rhGH from mammalian cell culture supernatant in a one-step process with high purity and yield. As the Gradiflow is directly scalable, this study also illustrates the potential for the inclusion of the Gradiflow into bioprocesses for the production of clinical grade rhGH and other therapeutic proteins. (C) 2003 Elsevier Science (USA). All rights reserved.

Hormone replacement therapy and breast cancer: estimate of risk: Education debate

The risk of breast cancer arises from a combination of genetic susceptibility and environmental factors. Recent studies show that type and duration of use of hormone replacement therapy affect a women's risk of developing breast cancer.1-7 The women's health initiative trial was stopped early because of excess adverse cardiovascular events and invasive breast cancer with oestrogen and progestogen.6 The publicity increased public awareness of the risks of hormone replacement therapy, and this was heightened by the publication of the million women study.2 However, the recently published oestrogen only arm of the women's health initiative trial suggests that this formulation may reduce the risk of breast cancer.8 To help make sense of the often confusing information,9 women and clinicians need individual rather than population risk data. We have produced estimates that can be used to calculate individual risk for women living up to the age of 79 and suggest the risk