Structural tendon changes in patients with acromegaly: assessment of Achilles tendon with sonoelastography.

AIMS: To describe the sonoelastographic appearance of the Achilles tendon in acromegalic patients and to determine whether the blood concentrations of growth hormone (GH) and insulin-like growth factor (IGF-1) are associated with the various sonographic elasticity types of Achilles tendons. MATERIAL AND METHODS: Eighty-four Achilles tendons of 42 acromegaly patients and 84 Achilles tendons of 42 healthy volunteers were assessed with sonoelastography. The tendons were classified into two main types according to the elasticity features: type 1 blue/green (hard tissue) and type 2 yellow/red within green (intermediate-soft tissue). Two subtypes of these types were also defined. According to the definition, the elasticity of the tissue was in a spectrum ranging from hard to soft as the type progressed from 1a to 2b. RESULTS: The mean thickness of Achilles tendons in patients with acromegaly was significantly higher compared with healthy Achilles tendons (5.1+/-0.7 mm vs. 4.4+/-0.5, p<0.001), and patients with active disease had thicker Achilles tendons (5.5+/-0.8 mm vs. 4.8+/-0.5 mm in inactive disease, p=0.003). A significantly higher proportion of acromegaly patients had type 2 sonoelastographic appearance of the Achilles tendon (124/252 third; 49.2% vs. 81/252 third; 32.1%, p=0.0001). Activity status of acromegaly and GH/IGF-I levels were similar in patients with different types of elasticity (p>0.05). CONCLUSIONS: Sonoelastography revealed structural changes in the tendinous tissue of patients with acromegaly, but it was not sensitive enough to reflect changes in the serum levels of GH/IGF-1.

Comprehensive biometric, biochemical and histopathological assessment of nutrient deficiencies in gilthead sea bream fed semi-purified diets.

Seven isoproteic and isolipidic semi-purified diets were formulated to assess specific nutrient deficiencies in sulphur amino acids (SAA), n-3 long-chain PUFA (n-3 LC-PUFA), phospholipids (PL), P, minerals (Min) and vitamins (Vit). The control diet (CTRL) contained these essential nutrients in adequate amounts. Each diet was allocated to triplicate groups of juvenile gilthead sea bream fed to satiety over an 11-week feeding trial period. Weight gain of n-3 LC-PUFA, P-Vit and PL-Min-SAA groups was 50, 60-75 and 80-85 % of the CTRL group, respectively. Fat retention was decreased by all nutrient deficiencies except by the Min diet. Strong effects on N retention were found in n-3 LC-PUFA and P fish. Combined anaemia and increased blood respiratory burst were observed in n-3 LC-PUFA fish. Hypoproteinaemia was found in SAA, n-3 LC-PUFA, PL and Vit fish. Derangements of lipid metabolism were also a common disorder, but the lipodystrophic phenotype of P fish was different from that of other groups. Changes in plasma levels of electrolytes (Ca, phosphate), metabolites (creatinine, choline) and enzyme activities (alkaline phosphatase) were related to specific nutrient deficiencies in PL, P, Min or Vit fish, whereas changes in circulating levels of growth hormone and insulin-like growth factor I primarily reflected the intensity of the nutritional stressor. Histopathological scoring of the liver and intestine segments showed specific nutrient-mediated changes in lipid cell vacuolisation, inflammation of intestinal submucosa, as well as the distribution and number of intestinal goblet and rodlet cells. These results contribute to define the normal range of variation for selected biometric, biochemical, haematological and histochemical markers.

Multiple protein biomarker assessment for recombinant bovine somatotropin (rbST) abuse in cattle.

Biomarker profiling, as a rapid screening approach for detection of hormone abuse, requires well selected candidate biomarkers and a thorough in vivo biomarker evaluation as previously done for detection of growth hormone doping in athletes. The bovine equivalent of growth hormone, called recombinant bovine somatotropin (rbST) is (il)legally administered to enhance milk production in dairy cows. In this study, first a generic sample pre-treatment and 4-plex flow cytometric immunoassay (FCIA) were developed for simultaneous measurement of four candidate biomarkers selected from literature: insulin-like growth factor 1 (IGF-1), its binding protein 2 (IGFBP2), osteocalcin and endogenously produced antibodies against rbST. Next, bovine serum samples from two extensive controlled rbST animal treatment studies were used for in vivo validation and biomarker evaluation. Finally, advanced statistic tools were tested for the assessment of biomarker combination quality aiming to correctly identify rbST-treated animals. The statistical prediction tool k-nearest neighbours using a combination of the biomarkers osteocalcin and endogenously produced antibodies against rbST proved to be very reliable and correctly predicted 95% of the treated samples starting from the second rbST injection until the end of the treatment period and even thereafter. With the same biomarker combination, only 12% of untreated animals appeared false-positive. This reliability meets the requirements of Commission Decision 2002/657/EC for screening methods in veterinary control. From the results of this multidisciplinary study, it is concluded that the osteocalcin - anti-rbST-antibodies combination represent fit-for-purpose biomarkers for screening of rbST abuse in dairy cattle and can be reliably measured in both the developed 4-plex FCIA as well as in a cost-effective 2-plex microsphere-based binding assay. This screening method can be incorporated in routine veterinary monitoring programmes: in the European Union for detection of rbST abuse and in the control of rbST-free dairy farms in the United States of America and other countries.

Assessment of endogenous growth hormone pulsatility in gelded yearling horses using deconvolution analysis.

HYPOTHESIS/OBJECTIVES: Defining normal Growth Hormone (GH) secretory dynamics in the horse is necessary to understand altered GH dynamics related to issues like welfare and disease. ANIMALS AND METHODS: Twelve healthy yearlings and two mature Standardbreds were used to quantify GH secretion. Endogenous GH half-life was determined after administration of 1.0 µg/kg BW GH releasing hormone (GHRH). Exogenous GH half-life was determined after administration of 20 µg/kg BW recombinant equine GH (reGH) with and without suppression of endogenous GH secretion by somatostatin infusion (50 µg/m(2)/h). Pulse detection algorithm (Cluster) as well as deconvolution analysis was used to quantify GH secretory dynamics based on GH concentration-time series sampled every 5 min from 22:00 till 06:00 h. In addition, reproducibility, impact of sampling frequency and influence of altering initial GH half-life on parameter estimates were studied. RESULTS: Mean endogenous GH half-life of 17.7 ± 4.4 (SD) min and mean exogenous half-life of 26.0 ± 2.9 min were found. The mean number of GH secretion peaks in 8 h was 12 ± 3.2. Ninety-nine percent of the total amount of GH secreted occurred in pulses, basal secretion was 0.012 ± 0.014 µg/L/min and half-life was 8.9 ± 2.6 min. Compared with a 5-min sampling frequency, 20- and 30-min sampling underestimated the number of secretory events by 45% and 100%, respectively. CONCLUSIONS: The deconvolution model used was valid to GH time series in Standardbreds. As in man, the equine pituitary gland secretes GH in volleys consisting of multiple secretory bursts, without measurable intervening tonic secretion. The required GH sampling frequency for the horse should be around 3 min. CLINICAL RELEVANCE: Defining normal GH secretory dynamics in the horse will make it possible to detect alterations in the GH axis due to pathophysiologic mechanisms as well as abuse of reGH.

Genetic variation of metabolite and hormone concentration in UK Holstein-Friesian calves and the genetic relationship with economically important traits.

The decline of dairy cattle fertility worldwide remains a major concern, with conception rates to first service commonly below 40%. The length and severity of negative energy balance postpartum are unfavorably correlated with fertility, suggesting that the length and severity of negative energy balance and fertility are linked via several hormones or metabolites. These compounds therefore have the potential to predict fertility at a genetic level. The addition of a predictor trait for fertility into present fertility indices would accelerate genetic gain, particularly if it was expressed before adulthood. The objective of this work was to estimate the genetic variation in several metabolites and hormones in calves, and to determine their genetic relationships with fertility and production through sire predicted transmitting abilities (PTA; sires of calves sampled). Circulating concentrations of free fatty acids (FFA), glucose, growth hormone (GH), insulin, and insulin-like growth factor 1 (IGF-1) in male and female UK Holstein-Friesian dairy calves (average age +/- SD; 126 +/- 12.7 d) were analyzed during 2 studies: data set 1 (n = 496 females; 1996-2001; 7 commercial dairy herds) and data set 2 (n = 326 females, n = 256 males; 2002-2006; multiple ovulation and embryo transfer breeding scheme). Univariate mixed models were fitted to the data using ASREML. Basal concentrations of FFA, glucose, GH, insulin and total IGF-1 were all moderately heritable in both sexes (heritability range +/- SE; 0.09 +/- 0.05 to 0.66 +/- 0.14). The sire PTA for protein percentage had significant regression coefficients and approximate genetic correlations with FFA and insulin, and the sire PTA for calving interval had significant regression coefficients and approximate genetic correlations with GH. Additive genetic variance seems responsible for a moderate proportion of the phenotypic variation in important metabolites and regulatory hormones in male and female UK Holstein-Friesian dairy calves, therefore supporting further investigation into their use as juvenile predictors for fertility in the mature female.

Diagnostic efficacy of 0, 30, 45, 60, 90 and 120 min growth hormone samples in insulin tolerance test: utility of growth hormone measurement at different time-points and a cost-effective analysis.

OBJECTIVE: To determine the utility of growth hormone (GH) measurement with the insulin tolerance test (ITT), and to carry out a cost-effective analysis of the diagnosis of GH deficiency. MATERIAL AND METHODS: Ninety-nine patients clinically suspected of GH deficiency were evaluated over a period of 14 months (January 2005 to April 2006). Post-insulin samples of GH and blood glucose (BG) samples were drawn at six different time-points. Serum GH levels of <10 microg/L (prepubertal) and <6.1 microg/L (adolescents) were taken as cut-off for the normal response. RESULTS: Ninety-nine ITTs were carried out during the study period, and GH levels were found to be deficient in 47 subjects. Specificities at different time-points were 0%, 54%, 77%, 62%, 39% and 23% for 0, 30, 45, 60, 90 and 120 min, respectively, in the prepubertal group, and 5%, 41%, 80%, 87%, 77% and 46% at the same time-points for the adolescent group. Accuracy was highest at 45 and 60 min in both the prepubertal and adolescent groups. The receiver operating characteristic curve showed that the highest area under the curve was found in samples drawn at 45, 60 and 90 min in both the prepubertal and adolescent groups. CONCLUSION: Our data suggest that 0, 45, 60 and 90 min samples are sufficient for diagnosing GH deficiency, which could lead to potential cost reductions of up to 29.8%.

Recombinant rat and mouse growth hormones: risk assessment of carcinogenic potential in 2-year bioassays in rats and mice.

Recombinant rat growth hormone (rrGH) and recombinant mouse growth hormone (rmGH) were developed to evaluate the potential carcinogenicity of each biologically active growth hormone (GH) as assessed in the respective species. Biological activities of rrGH and rmGH were demonstrated by showing an increase in body weight gain and serum levels of insulin-like growth factor-1 (IGF-1) in hypophysectomized rats receiving daily sc injections for 6 days. With the exception of pharmacologically mediated weight gain, rrGH and rmGH had no adverse effects in 5-week oral toxicity studies and no production of anti-recombinant GH antibodies. The high doses selected for the carcinogenicity studies provided systemic exposures of GH up to approximately 10-fold over basal levels. In the 105-week mouse carcinogenicity study, daily sc injections of rmGH at 0.1, 0.2, or 0.5 mg/kg/day were well tolerated and had no effects on survival or incidence of tumors. In the 106-week rat carcinogenicity study, daily sc injections of rrGH at 0.2, 0.4, or 0.8 mg/kg/day had a favorable effect on longevity in female rats administered 0.4 or 0.8 mg/kg/day, an increased weight gain in females and males, and no increase in the incidence of tumors. The absence of carcinogenic effects of recombinant GH administered daily for 2 years to rodents was consistent with publications of clinical experience, indicating a lack of convincing evidence for an increased risk of cancer in children receiving human recombinant GH replacement therapy.

Morphometric assessment of the impact of serum thymulin immunoneutralization on pituitary cell populations in peripubertal mice.

Thymulin is a thymic hormone involved in several aspects of intra- and extrathymic T-cell differentiation. Thymulin also possesses hypophysiotropic activity which suggests that this metallopeptide may play an important role in thymus-pituitary communication, particularly during early life. The aim of the present study was to evaluate the impact of serum thymulin suppression from birth to peripuberty on the morphology of different pituitary cell populations in prepubertal C57Bl/6 mice. Animals were submitted to immunoneutralization of circulating thymulin from postnatal day 1 to the end of the study (age 32 days). From their 1st day of life, the animals were submitted to a protocol of intraperitoneal injections of rabbit anti-thymulin serum (alpha-FTS) and normal rabbit serum (NRS) in the controls. On their 33rd day of life, the animals were killed and their pituitaries were immediately dissected, fixed and immunostained using the EnVision system with primary antibodies against growth hormone, thyrotropin, corticotropin, gonadotropins and prolactin. Morphometry was performed by means of an image analysis system. The following parameters were calculated: volume density = Sigma cell area/reference area (RA); cell density (CD) = number of cells/RA, and cell size (expressed in microm2). Serum thymulin was measured by a rosette bioassay while pituitary hormones were assayed by radioimmunoassay. Serum prolactin, luteinizing hormone, follicle-stimulating hormone, growth hormone and thyroid-stimulating hormone were significantly lower in the alpha-FTS animals of either sex compared with the corresponding NRS counterparts. The somatotrope, lactotrope and corticotrope populations showed a significant decrease in CD, while cell hypertrophy was observed in some of the pituitary cell populations of the alpha-FTS group compared to the NRS group. In the alpha-FTS group, there were sex differences in the morphometric changes observed. Our results suggest that serum thymulin plays a significant role during early life in the postnatal maturation of endocrine cells of the mouse anterior pituitary gland.

Regulating of growth hormone sensitivity by sex steroids: implications for therapy.

Growth hormone (GH) is an important regulator of body composition, reducing body fat by stimulating fat oxidation and enhancing lean body mass by stimulating protein accretion. The emergence of differences in body composition between the sexes during puberty suggests sex steroids modulate the action of GH. Work from our laboratory have investigated the influence of estrogens and androgens on the metabolic actions of GH in human adults. The liver is an important site of physiological interaction as it is a sex steroid responsive organ and a major target of GH action. Estrogen, when administered orally impairs the GH-regulated endocrine and metabolic function of the liver via a first-pass effect. It reduces circulating IGF-I, fat oxidation and protein synthesis, contributing to a loss of lean and a gain of fat mass. These effects occur in normal and in GH-deficient women and are avoided by transdermal administration of physiological doses of estrogen. In contrast, studies in hypopituitary men indicate that testosterone enhances the metabolic effects of GH. Testosterone alone stimulates fat oxidation and protein synthesis, both of which are enhanced by GH. Studies in GH deficiency adults have consistently reported women to be less sensitive to GH than men. In summary, estrogens and androgens exert divergent effects on the action of GH. The results provide an explanation for sexual dimorphism in body composition in adults and the gender-related response to GH replacement in hypopituitary subjects. In the management of hypopituitarism, estrogens should be administered by the parenteral route in women and testosterone be replaced in men to optimize the benefits of GH replacement.

Assessment of GH reserve before and after successful treatment of adult patients with Cushing's syndrome.

OBJECTIVE: Several studies have demonstrated impaired GH secretion in patients with active Cushing's syndrome (CS). It has been suggested that persistence of GH deficiency, despite treatment of cortisol excess, may delay the recovery of these patients and therefore temporary treatment with GH may have some benefit. However, the time course of restoration of GH secretion after successful treatment of CS has only been investigated in a limited number of mostly paediatric reports. The aim of the present study was the evaluation of GH reserve in adult patients with CS before and after correction of cortisol excess. DESIGN AND PATIENTS: Sixteen patients (12 females, four males) with CS aged 44.7 +/- 5.05 years were recruited. These included seven patients with Cushing's disease, four patients with ectopic ACTH secretion and five patients with adrenal adenoma. All patients were evaluated before any therapeutic intervention. Twelve patients were successfully treated following appropriate surgery and these were further studied. The combined pyridostigmine/GHRH test was used to assess GH reserve in these patients. In a proportion of cases an insulin tolerance test (ITT) was also used. RESULTS: Before any therapeutic intervention, an impaired GH response to PD/GHRH was noted in all patients. Restoration of GH response at 6 months was observed in six patients (50%); at 12 months in two; at 18 months in one patient. Two of the patients with no restoration of GH response at 12 months did not accept further investigation. Only one patient did not achieve an adequate GH response even when tested 30 months following cure of CS. Restoration of GH reserve was more commonly observed in those patients in whom there was recovery of the HPA axis. There was a good correlation between peak GH levels to PD + GHRH and ITT. No statistically significant difference was revealed in IGF-I levels between pre- and post-treatment evaluation. CONCLUSIONS: Adult patients with active Cushing's syndrome demonstrate a profound suppression of stimulated GH secretion. In the majority of these patients the disruption of GH secretion is normalized within a year after successful treatment of endogenous cortisol excess.

Development and validation of a simple sensitive enzyme immunoassay (EIA) for GH determination in buffalo plasma.

A simple and highly sensitive enzymeimmunoassay (EIA) for GH determination in buffalo plasma on microtitreplates using biotin-streptavidin amplification system and the second antibody coating was developed. Biotin was coupled to GH and used to bridge between streptavidin-peroxidase and immobilized antiserum in competitive assay. The EIA was carried out directly in 100 microL buffalo plasma. The GH standards ranging from 0.05 ng/well/100 microL to 12.8 ng/well/100 microL were prepared in hormone free plasma collected from an aged (> 15 years) senile buffalo. The sensitivity of the EIA procedure was 50 pg/well GH. which corresponded to 0.50 ng/mL plasma; the 50% relative binding sensitivity was seen at 800 pg/well/100 microL. Plasma volumes for the EIA, viz., 25, 50, and 100 microL did not influence the shape of standard curve, even though a slight drop in the OD450 was seen with higher plasma volumes. For the biological validation of the assay, 12 Murrah buffalo calves were used. Six of these were administered synthetic bovine growth hormone-releasing factor (10 microg/100 kg body weight, i.v., and the remaining six animals were administered sterile normal saline and kept as controls. Jugular blood samples were collected at -60, -45, -30, -15, -10, -5, 5, 10, 15, and 30 min and, thereafter, at an interval of 15 min using an indwelling jugular catheter, beginning 1 h prior to GRF injection up to 8 h post treatment. In all animals, a peak of GH was recorded within 5 to 20 min of GRF administration, which confirms the biological validation of the EIA. To confirm homogeneity of buffalo GH with bovine GH, a parallelism test was conducted between the buffer standard curve of bovine GH and GH measured from serial dilution of buffalo plasma containing a high level of endogenous growth hormone.

Development of an enzyme-linked immunosorbent assay for the measurement of plasma growth hormone (GH) levels in channel catfish (Ictalurus punctatus): assessment of environmental salinity and GH secretogogues on plasma GH levels.

We report the development of a sensitive, and specific, competitive, antigen-capture enzyme-linked immunosorbent assay for the measurement of channel catfish (Ictalurus punctatus) growth hormone (cfGH). The detection limit of the assay (90% binding) was 2.0ng/ml and the ED(50) value (standard curve range 150-0.59 ng/ml) was 67.3 ng/ml. Recovery of cfGH-spiked plasma samples was determined to be 102%. Dose-response inhibition curves using serially diluted pituitary homogenates and plasma samples consistently showed parallelism with the standard curves using purified cfGH. The GH antibody (rabbit anti-catfish GH) specificity was demonstrated in competitive binding curves employing heterologous hormones and purified channel catfish prolactin (cfPRL). These studies show that there was no significant (0.006%) binding of cfPRL (competitive inhibition of cfGH binding), or heterologous hormones, within the working range of the assay. To physiologically validate the assay, catfish were injected (100 microg/g body weight, 3 injections every 5 days) with either bovine GHRH(1-29)-amide or the synthetic hexapeptide GHRP-2 (KP-102: D-Ala-D-beta-Nal-Ala-Trp-D-Phe-Lys-NH(2)) suspended in corn oil. Following the last injection, half of the animals were sampled for plasma and the remaining transferred from fresh water (FW) to 12 ppt seawater (BW: brackish water). Twenty-four hours after transfer to BW, animals were again sampled for plasma. Plasma GH levels were significantly (p<0.001) elevated in all the BW groups (control, KP-102, and bGHRH), compared with the FW (fresh water) groups. In addition, plasma GH levels were significantly (p<0.001) elevated by treatment with either of the GH secretogogues, KP-102 or bGHRH. Our findings demonstrate that two regulatory mechanisms of GH elevation, one which is seen in euryhaline teleosts (salinity-induced GH levels) and another, which has been recently described in teleosts (GHRP-induced GH levels), are present in the stenohaline channel catfish.

An audit of the laboratory interpretation of growth hormone response to insulin-induced hypoglycaemia in the assessment of short stature in children.

INTRODUCTION: Measurement of serum growth hormone (GH) concentration in response to insulin-induced hypoglycaemia remains an important investigation in the assessment of pituitary disease. METHODS: In this audit, laboratories were presented with aliquots of sera with GH concentrations likely to be found in an insulin stress test (IST). They were invited to analyse the specimens for GH and comment on their results. RESULTS AND DISCUSSION: A number of laboratories appeared to be using out-of-consensus cut-off concentrations that sometimes were unrelated to the bias of their GH assay. The specimens were chosen to mimic those seen in an IST that was clearly not indicative of GH deficiency, so there was reasonable consensus in the interpretation of results. However, five laboratories (9.6%) did indicate that their results were equivocal.

Height velocity and IGF-I assessment in the diagnosis of childhood onset GH insufficiency: do we still need a second GH stimulation test?

OBJECTIVE: The diagnosis of GH insufficiency (GHI) in childhood is not straightforward. Our aim was to test the sensitivity and specificity of height velocity (HV), IGF-I, IGFBP-3 and GH stimulation tests alone or in combination in the diagnosis of GHI. DESIGN: A retrospective review of patients with GHI and idiopathic short stature (ISS) diagnosed in our centre and followed up to the completion of linear growth. PATIENTS: Thirty-three GHI children and 56 children with ISS were evaluated. GHI diagnosis was based on fulfilment of anthropometric, endocrine and neuroradiological criteria: stature < or = -2 z-score, delayed bone age (at least 1 year), GH peak response to at least two different provocative tests < 10 micro g/l (20 mU/l), brain MRI positive for hypothalamus-pituitary abnormalities, catch-up growth during the first year of GH replacement therapy > or = 75th centile, peak GH response to a third provocative test after growth completion < 10 micro g/l (20 mU/l). Children with anthropometry resembling that of GHI but with peak GH responses > 10 micro g/l (20 mU/l) were diagnosed as ISS. MEASUREMENTS: All subjects underwent standard anthropometry. GH secretory status was assessed by clonidine, arginine and GHRH plus arginine stimulation tests. IGF-I and IGFBP-3 circulating levels were measured by immunoradiometric assay (IRMA). The following cut-off values were chosen to discriminate between GHI and nonGHI short children: HV < 25th centile over the 6-12 months prior to the initiation of GH therapy, peak GH responses < 10 or < 7 micro g/l (< 20 or < 14 mU/l) and IGF-I and IGFBP-3-values < -1.9 z-score. Sensitivity (true positive ratio) and specificity (true negative ratio) were evaluated. RESULTS: Taking 10 micro g/l (20 mU/l) as the cut-off value, sensitivity was 100% and specificity 57% for GH provocative tests, whereas taking 7 as the cut-off value, sensitivity was 66% and specificity rose to 78%. Sensitivity was 73% for IGF-I and 30% for IGFBP-3 measurement, whilst specificity was 95% for IGF-I and 98% for IGFBP-3 evaluation. HV assessment revealed a sensitivity of 82% and a specificity of 43%. When HV and IGF-I evaluations were used in combination, sensitivity reached 95% and specificity 96%. When both HV and IGF-I are normal (26% of our subjects) GHI may be ruled out, whereas when both the indices are subnormal (23%) GHI is so highly likely that the child may undergo only one GH provocative test and brain MRI and, thereafter, may begin GH therapy without any further test. In case of discrepancy, when IGF-I is normal and HV < 25th centile (44% of children), due to the relatively low sensitivity of IGF-I assessment and low specificity of HV, the patient should undergo GH tests and brain MRI. Finally, in the rare case of HV > 25th centile and subnormal IGF-I-values (7%), due to the high specificity of IGF-I measurement, the child should undergo one provocative test and brain MRI for the high suspicion of GHI. CONCLUSIONS: Our results suggest that a simple assessment of HV and basal IGF-I may exclude or, in association with only one stimulation test, confirm the diagnosis of GH insufficiency in more than half of patients with short stature.

Growth hormone abuse in the horse: preliminary assessment of a mass spectrometric procedure for IGF-1 identification and quantitation.

Previous studies have shown that insulin-like growth factor 1 (IGF-1) is a promising marker for the detection of growth hormone (GH) abuse in the horse. The significant increases observed with GH administration in comparison to natural levels imply the possibility of setting a threshold level for IGF-1 that would be indicative of GH abuse. Although an immunoradiometric assay (IRMA) has been identified as a reliable screening method, a more specific IGF-1 quantification method needs to be developed for the prosecution of GH abuse by horseracing authorities. This study describes such an HPLC electrospray mass spectrometry (LC/ESI-MS) method that was developed and then assessed for the specific analysis of IGF-1 at the low levels encountered in serum. The structural identity of IGF-1 was confirmed by endoproteinase Asp-N digestion followed by LC/MS and LC/MS/MS characterisation. This was followed by quantification of IGF-1 as the intact molecule against an internal standard.

Reproducibility of the insulin tolerance test (ITT) for assessment of growth hormone and cortisol secretion in normal and hypopituitary adult men.

OBJECTIVE: The within subject variability of the insulin tolerance test (ITT) for assessment of growth hormone (GH) status and cortisol reserve has rarely been examined, particularly in patients with hypopituitarism. This becomes important when biochemical criteria are used to determine which adults with hypopituitarism should receive GH and/or cortisol replacement. In the present study we assessed the reproducibility of GH and cortisol responses in repeated ITTs. Baseline insulin-like growth factor 1 (IGF-1) levels were also assessed for reproducibility on each occasion. DESIGN AND PATIENTS: Three consecutive ITTs were performed in seven normal adult men (ages 22-27 years) and two ITTs in 11 men with hypopituitarism and suspected GH deficiency (ages 23-48 years). MEASUREMENTS: Serum GH and IGF-1 were measured by immunoradiometric and cortisol by immunofluorimetric assays. RESULTS: In normal men group peak GH responses did not differ between the three tests. There was no correlation between individual peak values. The within subject peak GH variability was between 4.6 and 59.3%, and the overall variability in 21 tests was 35%. The lowest peak GH concentration was 70 mU/l (27 microg/l). All hypopituitary men had severe GH deficiency (all peak GH concentrations < 4 mU/l (1.5 microg/l) in both tests). There was a highly significant correlation between individual peak GH values (r = 0.95, P < 0.0001). Basal IGF-1-values in normal and hypopituitary men were highly correlated between tests (r = 0.98, P < 0.0001). The overall within subject variability of IGF-1-values was 11.9% in normal and 22.7% in hypopituitary men. In normal men group peak cortisol responses were not different between the three tests. There was a good correlation between individual peak cortisol responses in the three ITTs. The within subject peak cortisol variability (median 8.3%; range 0.7-21.5%) was significantly less than that of GH (P < 0.03) in two of three test comparisons. In hypopituitary men the within subject peak cortisol variability (median 41.6%; range 3.5-92.7%) was significantly greater (P < 0.001) than in normal men. All patients were correctly classified as cortisol deficient or normal in both ITTs. CONCLUSION: The cortisol response to repeated hypoglycaemia is very reproducible in normal men but the GH response is less so. In hypopituitary men the reproducibility of the GH response is good while that of the cortisol response is poor. However, a single ITT did not misclassify hypopituitary patients who are severely GH and/or ACTH deficient and was therefore adequate for clinical decisions regarding GH and/or cortisol replacement. Nevertheless, it remains possible that a single ITT could misclassify some hypopituitary patients with partial GH or ACTH deficiency.

Treatment of Chinese acromegaly with a combination of bromocriptine and octreotide.

BACKGROUND: Good results have been reported with combined use of octreotide and bromocriptine in acromegalic Caucasians. Data concerning the efficacy and tolerability of this combination treatment in Chinese acromegalic patients are scanty. AIM: The aim of this study was to assess the efficacy and tolerability of combined therapy using bromocriptine and octreotide in the treatment of acromegaly in Chinese patients and to compare the cost-effectiveness of various regimes. METHODS: Sixteen Chinese acromegalic patients with growth hormone (GH) concentration not suppressible to below 5 mU/L (2 microg/L) during an extended OGTT were recruited to undergo four phases of the study. During the study period, the patients were given bromocriptine alone, bromocriptine and low dose octreotide, bromocriptine and medium dose octreotide, and medium dose octreotide alone. Plasma concentrations of GH and insulin-like growth factor-1 (IGF-1) were measured before and after the completion of each phase. RESULTS: The number of patients reaching target GH concentrations was significantly higher when treated with octreotide compared to baseline (p<0.05). Bromocriptine alone had a significant effect but not to the extent of octreotide alone. A combination of low dose octreotide and bromocriptine is as efficacious in the treatment of acromegaly as high dose octreotide. None of the patients suffered from serious adverse effects. CONCLUSION: The results confirmed the usefulness and tolerability of bromocriptine and octreotide in Chinese acromegalics. The most cost-effective regime in this study was a combination of low dose octreotide and bromocriptine.

GH response to exercise: assessment of the pituitary refractory period, and relationship with circulating components of the GH-IGF-I axis in adolescent females.

The concept of pituitary refractory period for GH secretion has been previously described. To measure the length of this refractory period we performed an exercise provocation test for GH secretion immediately following multiple overnight GH blood sampling. In addition, we correlated the magnitude of the GH response to a single exercise input with mean overnight GH, IGF-I and circulating IGFBP levels. 23 healthy adolescent females (15-17 yr) performed 10-min constant cycle ergometry at a power normalized to each subject's aerobic and anaerobic capacity. GH was measured every 10 min starting 10 min before exercise and then for 60 min after the exercise bout. Mean nocturnal GH was calculated from overnight values obtained every 20 min over a 12-h period. Pre-exercise GHBP, IGF-I and IGFBPs 1-5 were assessed using standard techniques. In five subjects, a spontaneous GH peak had preceded the exercise test by 1 hour or less, and no response to exercise was found. In the remaining 18 subjects, a GH peak (6.8 +/- 1.3 ng/ml, p < 0.0001) was observed at 32 +/- 4 min after the onset of exercise. The GH response to exercise was not correlated with fitness, mean GH or IGF-I but was correlated with IGFBP-3 (r = 0.65, p < 0.05). Spontaneous GH pulses may acutely render the pituitary refractory to exercise stimuli. The length of this refractory period is approximately 1 hour. The data corroborate the idea that while relationships exist among the various components of the GH-IGF-I axis, no single factor identified to date fully reflects GH-IGF-I "tone".