Leptin concentrations in maternal serum and cord blood: relationship to maternal anthropometry and fetal growth.

OBJECTIVE: To determine 1. the relationship between maternal serum leptin concentrations and maternal anthropometry and 2. the relationship between cord serum leptin concentrations at birth and neonatal anthropometry. DESIGN: Prospective cohort study of fetal growth in low-risk pregnancies. SETTING: University teaching hospital. SAMPLE: Thirty-nine women and their babies taking part in a fetal growth study. METHODS: Blood was taken from the women between 10-20 weeks of gestation and from the umbilical cord of their babies at delivery. Serum leptin was measured by radio-immunoassay. Maternal anthropometric measurements were recorded at booking. Neonatal anthropometric measurements were recorded within 48 hours after delivery. Linear regression analysis was used to explore the relationship between serum leptin concentrations and anthropometric measures and multiple regression analysis then applied to determine which variables remained independently associated with leptin. RESULTS: The median (range) leptin concentration in maternal serum was 11.8 ng/mL (1.7-39.7) and in cord blood was 4.2 ng/mL (0.6-21.4). Maternal leptin levels correlated with maternal weight, body mass index, midarm circumference and skinfold thickness, but not with birthweight, placental weight or maternal height. Body mass index and midarm circumference remained significant after multiple regression analysis. Cord leptin levels correlated with birthweight, birthlength, placental weight and skinfold thickness but not with ponderal index. Birthweight and subscapular skinfold thickness remained significant after multiple regression analysis. Cord leptin concentrations did not correlate with maternal leptin concentrations. CONCLUSIONS: We suggest that there are very strong associations between maternal leptin and maternal adiposity in pregnancy, and between cord leptin at delivery and birthweight, as well as other anthropometric markers of fetal growth.

Ontogeny of serum leptin concentrations in the human.

OBJECTIVE: Serum leptin concentrations reflect the fat mass of an individual. Fetal growth is rapid and it might be expected that major changes in circulating leptin concentrations in the fetus and neonate take place. We have studied the ontogeny of serum leptin concentrations in cord blood samples obtained by cordocentesis between 14 and 32 weeks of gestation and in samples obtained at term. PATIENTS: Cordocentesis samples from 10 appropriately grown for gestational age (AGA) and 10 intrauterine growth restricted (IUGR) fetuses. The results were compared with cord serum leptin concentrations obtained in 39 term healthy pregnancies. RESULTS: In the AGA and term pregnancies serum leptin concentrations changed little up until 38 weeks of gestation when there was an increase in concentration (Pre 38 weeks 3.5 microg/l (SEM 0.3); Post 38 weeks 5. 6 microg/l (SEM 0.7): Mann Whitney P = 0.03). Serum leptin concentrations were similar in the AGA and IUGR fetuses (AGA 5.9 microg/l (SEM 3.0); IUGR 4.2 microg/l (SEM 1.5): Mann-Whitney P = ns). Serum leptin was strongly related to birth weight (r = 0.58; P < 0.001) and birth length (r = 0.32; P = 0.05) in the term babies but not in the AGA or IUGR groups. CONCLUSION: Serum leptin concentrations in the fetus appear to be independent of gestational age and only rise towards the end of gestation. This late change probably reflects the changes in the accretion of body fat. There appears to be little difference in serum leptin concentrations between AGA and IUGR fetuses.

A sexually dimorphic pattern of growth hormone secretion in the elderly.

In rodents, the sexually dimorphic pattern of pulsatile GH secretion is an important determinant of growth, liver enzyme function and insulin-like growth factor I (IGF-I) expression. Whether this difference is present in humans at different ages is unclear. We studied GH secretory patterns in the elderly by constructing 24-h serum GH profiles in 45 male and 38 female (age, 59.4-73.0 yr) volunteers and related patterns to IGF-I, IGF-binding protein-3 (IGFBP-3), and GH-binding protein levels; body mass index; and waist/hip ratio. Serum GH concentrations were measured in samples drawn at 20-min intervals and analyzed using a sensitive chemiluminescent assay (Nichols Institute Diagnostics: sensitivity, 0.036 mU/L). The 24-h serum GH profiles were analyzed using a concentration distribution method to determine GH peak and trough levels, spectral analysis, and assessment of serial irregularity by approximate entropy (ApEn). There was a highly significant difference in mean 24-h serum GH concentrations in females compared to males (males, 0.88 mU/L; females, 1.31 mU/L; P = 0.009) as a result of significantly higher trough GH levels (males, 0.04 mU/L; females, 0.16 mU/L; P < 0.001). Peak values were not significantly different. Serum IGF-I levels were significantly higher in males (males, 162.4 ng/mL; females, 87.8 ng/ mL; P < 0.001). Peak GH values were related to serum IGF-I levels (males: r = 0.39; P = 0.009; females: r = 0.5; P = 0.002), whereas trough GH levels were not. IGFBP-3 levels were similar and related to GH peaks only in males (r = 0.32; P = 0.03). GH was secreted with a dominant periodicity of 200 min in males and 280 min in females (P < 0.025). The proportion of time taken up by regular oscillatory activity was less in females (females, 11.1%; males, 14.7%; P = 0.01). GH secretion assessed by ApEn was more disordered in females (males, 0.60; females, 0.81; P < 0.001), and increasing disorder was associated with lower IGF-I levels. Body mass index was negatively related to GH in both sexes. In males, trough values were the major determinant (r = -0.31; P = 0.04), whereas in females, the peak value was the major determinant (r = 0.35; P = 0.04). Trough GH levels were inversely related in both sexes to waist/hip ratio (males: r = -0.40; P = 0.006; females: r = -0.44; P = 0.006) and to increasing secretory disorder (ApEn; r = -0.46; P < 0.001). These data demonstrate a sexually dimorphic pattern of GH secretion in the elderly.

Tests of adrenal insufficiency.

AIM: In suspected adrenal insufficiency, the ideal test for assessing the hypothalamo-pituitary-adrenal axis is controversial. Therefore, three tests were compared in patients presenting with symptoms suggestive of adrenal insufficiency. METHOD: Responses to the standard short Synacthen test (SSST), the low dose Synacthen test (LDST), and the 08:00 hour serum cortisol concentration were measured in 32 patients. A normal response to the synacthen test was defined as a peak serum cortisol of >/= 500 nmol/l and/or incremental concentration of >/= 200 nmol/l. The sensitivity and specificity of the 08:00 hour serum cortisol concentration compared with other tests was calculated. RESULTS: Three patients had neither an adequate peak nor increment after the SSST and LDST. All had a serum 08:00 hour cortisol concentration of < 200 nmol/l. Eight patients had abnormal responses by both criteria to the LDST but had normal responses to the SSST. Three reported amelioration of their symptoms on hydrocortisone replacement. Twenty one patients had a normal response to both tests (of these, 14 achieved adequate peak and increment after both tests and seven did not have an adequate peak after the LDST but had a normal increment). The lowest 08:00 hour serum cortisol concentration above which patients achieved normal responses to both the LDST and SSST was 500 nmol/l. At this cut off value (compared with the LDST), the serum 08:00 hour cortisol concentration had a sensitivity of 100% but specificity was only 33%. CONCLUSION: The LDST revealed mild degrees of adrenal insufficiency not detected by the SSST. The value of a single 08:00 hour serum cortisol concentration is limited.

Cortisol, androstenedione (A4), dehydroepiandrosterone sulphate (DHEAS) and 17 hydroxyprogesterone (17OHP) responses to low doses of (1-24)ACTH.

The dose of 250 microg used in the standard short synacthen test is supraphysiological and lower doses may provide a more sensitive test. We examined steroid responses to 125ng/m2, 250ng/m2 and 500 ng/m2 (1-24)ACTH in 6 normal males, looking at effects of dose and the within- and between-subject coefficients of variation (CV). Subjects were given each dose 3 times, blood samples were taken at 10 minute intervals. There was a dose response relationship between dose of (1-24)ACTH and peak values for cortisol and 17OHP (p<0.05). There was no difference between peaks of A4 at different doses and no clear peaks were reached for DHEAS. 86% of the peaks for 17OHP, 63% for A4 and 25% for cortisol were at 10 minutes and 14%, 29% and 65% respectively at 20 mins (p=0.001). Within-subject CV for cortisol was 12.6% and between subject 10.1%. Tests of adrenal function using low doses of (1-24)ACTH have acceptable between- and within-subject CV for peak values with a dose as low as 125 ng/m2 (1-24)ACTH. Protocols for low dose synacthen tests, with traditional sampling at zero, 30 and 60 minutes or even as shown here at 10 minute intervals, fail to fully define the changes in steroid levels following adrenal stimulation. More frequent blood sampling will be needed to accurately detect peak levels in particular of 17OHP and A4.

Apal polymorphism in insulin-like growth factor II (IGF2) gene and weight in middle-aged males.

In 1474 healthy Caucasoid men aged 45-65 y, insulin-like growth factor II (IGF2) Apal AA homozygotes showed a mean body weight 4 kg lower than Apal GG homozygotes (77.6 +/- 10.9 kg vs 81.6 +/- 11.5 kg, P = 0.003) with heterozygotes (GA) intermediate (80.1 +/- 11.9 kg). The mean serum IGF-II concentration in 44 Apal AA individuals was significantly higher than in 48 Apal GG individuals (683.3 +/- 146.9 ng/ml vs 614.0 +/- 124.0 ng/ml, P = 0.01). An INS Pstl polymorphism showed no association with weight and it was also found to be in minimal linkage disequilibrium with the IGF2 Apal site (coefficient 0.016). The IGF2 Apal AA genotype is therefore associated with lower mean body weight but higher serum IGF-II concentrations than the GG genotype. Apal GG homozygotes incur a 1.67-fold risk of pathological Body Mass index (BMI) (> 30 kg/m2) compared with AA homozygotes.

Peak and trough growth hormone concentrations have different associations with the insulin-like growth factor axis, body composition, and metabolic parameters.

GH is secreted in a pulsatile fashion, promoting growth and anabolism. The components of the pulsatile signal involved in these diverse effects are unclear. We constructed (20-min sampling interval) and analyzed 24-h serum GH profiles in 45 adult male volunteers, 59.4-69.9 yr old, body mass index (BMI) 21.9-36.5 Kg/m2, using Fourier transformation and a concentration distribution analysis that determines the concentration at or below which the serum GH concentrations in the 24-h profile spend a percentage of the total time. The observed concentrations (OC) below which 95% and 5% of the values in the time series lie [lsb]OC95 (peaks) and OC5 (troughs)] and mean 24-h serum GH concentrations were related to measures of the insulin-like growth factor (IGF) family, parameters of body composition, fasting insulin and cholesterol measures, and GH-binding protein concentrations. Mean 24-h serum GH concentrations ranged between 0.19 and 2.15 mU/L (1 microgram/L = 2.6 mU/L). Pulse periodicity was between 180 and 200 min. There was a positive relationship between peak GH levels and serum IGF-1 and IGFBP-3 levels (r = 0.39; P = 0.009 and r = 0.32; P = 0.03, respectively). GH trough levels were unrelated to these measures of the IGF family. In contrast, GH troughs were related inversely to BMI (r = -0.31; P = 0.04) and waist-hip ratio (r = -0.4; P = 0.006). Peak GH levels were not related to these measures. Factors known to influence these measures, fasting insulin concentration, or cortisol secretion did not alter the trough GH relationship in multiple regression analysis. All GH parameters were related inversely to fasting insulin concentration. Although GH parameters were related inversely to cholesterol and low-density lipoprotein-cholesterol, this effect disappeared when age and fasting insulin levels were introduced into the regression. GH-binding protein levels related most strongly to BMI (r = 0.60; P < 0.001), with no effect of any GH parameter observed in multiple regression analysis. These results suggest that the peak values of a GH concentration profile may influence the IGF axis, whereas trough values may influence body composition and metabolic parameters of GH action.

Evaluation of growth hormone (GH) responses to pulsed GH-releasing hormone administration using the MTT-ESTA bioassay.

We compared the immunoactivity of human growth hormone (hGH) with its bioactivity after stimulation of hGH release into the circulation by the administration of growth hormone-releasing hormone [GHRH(1-29)-NH2] according to a pre-determined protocol to four normal adult volunteers. We used the Hybritech immunoradiometric assay to measure the immunoactive GH concentrations. Bioactive GH concentrations were measured using the highly quantitative and precise eluted stain bioassay system (ESTA). The high sample capacity of the ESTA bioassay permitted us to monitor the bioactivities in closely timed sequential samples, and in far greater detail than has previously been possible. Two pulses of GHRH(1-29)-NH2 were administered intravenously to the four adult male volunteers (aged 24-37 years) on a weekly basis over a 4-week period. Two different doses of GHRH(1-29)-NH2 (0.1 and 1.0 micrograms/kg) were tested. These were separated by specified time intervals (60 or 120 min). Responses in the four individuals were variable. However, although the immuno- and bioactivities generally agreed well, there was a systematic and progressive increase in the bioactivity/immunoactivity (B/I) ratios as half of the response peaks were approached. After these peak concentrations, the B/I ratios subsequently returned to values that were close to unity. The enhanced bioactivity of the peak samples from the two volunteers in whom the largest magnitudes of response were observed was found to be labile after long-term storage at -20 degrees C. We suggest that the preferential rise in GH bioactivity, as opposed to immunoactivity, in response to GHRH(1-29)-NH2 was due to progressive changes in the concentrations of isoforms of GH that are not detectable in the Hybritech immunoassay.

An investigation into the lability of the bioactivity of human growth hormone using the ESTA bioassay.

We compared the bioactivity attributable to human growth hormone (hGH) in serum samples, determined at the time of their collection, with that after storage for 2-18 months at -20 degrees C. The samples were obtained from volunteers and patients who underwent provocative tests of hGH secretion, and the bioactivity was determined in the ESTA bioassay, which is based upon the use of Nb2 cells. We report that, in some subjects, the bioactivity of samples collected at the response peaks deteriorated on storage for as little as 2 months. The decrease in hGH bioactivity was systematic in that it consistently declined so as to approach the values initially determined by an immunoassay (Hybritech IRMA). This differential lability was a characteristic of the peak samples, and was not observed for either samples collected before and after the peaks of hGH secretion or for purified preparations of hGH which were subjected to a range of freeze/thaw and storage regimens. We suggest that this unusual lability is indicative of transient shifts in the spectrum of the variants of hGH which are present in the circulation following stimulation by provocative agents. This study emphasises the need to minimise the risk of introducing storage artefacts in investigations into the responses of hGH to provocation.

GH responses to intravenous bolus infusions of GH releasing hormone and GH releasing peptide 2 separately and in combination in adult volunteers.

OBJECTIVE: Synthetic growth hormone releasing peptides (GHRP) have potent GH-releasing activity in vivo and in vitro. The nature of the interaction of GHRP and naturally occurring GH releasing hormone (GHRH) is still far from clear. We investigated GH release in response to individual peptide doses or combined doses of GHRH1-29NH2 and GHRP-2, a novel GH-releasing peptide, in normal adults. DESIGN: Subjects underwent three tests in a randomized order: (1) i.v. bolus of GHRH1-29NH2 (1 microgram/kg BW), (2) i.v. bolus of GHRP-2 (1 microgram/kg BW), (3) i.v. bolus of GHRH1-29NH2 combined with GHRP-2 (same dosages). SUBJECTS: Eight healthy non-obese male volunteers, aged 25-34 years. MEASUREMENTS: Serum GH concentrations were measured by IRMA at -15, 0, +10, 20, 30, 45, 60, 75, 90 and 120 minutes after the boluses. RESULTS: Peak GH levels in response to GHRH1-29NH2, GHRP-2 and the combined GHRH1-29NH2 and GHRP-2 administrations were observed between 20 and 45 minutes. Peak GH levels at 30 minutes were 32.8 +/- 27.3 (mean +/- SD), 109.7 +/- 56.1 and 140.9 +/- 80.6 mU/l, respectively. The area under the curve for GH levels (GH AUC) calculated for the first 90 minutes after the GHRH1-29NH2 test (2061.2 +/- 1601.9 mU/lmin) was significantly lower than those after GHRP-2 (6205.1 +/- 3216.9 mU/lmin) and the combined GHRH1-29NH2 and GHRP-2 challenge (9788.3 +/- 5530.4 mU/lmin) (P = 0.0003 and P = 0.00005, respectively; paired Student's t-test for log transformed data). Although the GH AUC of the GHRP-2 test and the combined GHRH1-29NH2 and GHRP-2 test differed significantly (P = 0.016, t-test), the latter was not significantly different from the sum of the GH AUCs of each subject after the separate tests. CONCLUSION: Although the GH releasing potency of GHRP-2 significantly exceeded that of GHRH1-29NH2, we were not able to demonstrate synergy between the two substances. It is possible that GHRP-2 given in our study GHRP-2 significantly exceeded that of GHRH1-29NH2, we were not able to demonstrate synergy between the two substances. It is possible that GHRP-2 given in our study in higher molar quantities than GHRH1-29NH2 masked the effect of the latter.

The measurement of growth hormone bioactivity in patient serum using an eluted stain assay.

We have adapted the MTT-ESTA bioassay for human GH (hGH) to measure the lactogenic bioactivity of the hormone in human serum. This highly quantitative in vitro colorimetric bioassay is based upon the reduction of a tetrazolium salt, 3-[4,5-dimethyl-thiazol-2-yl]2,5-diphenyl tetrazolium bromide (MTT), to its formazan by lactogen-activated Nb2 cells. Relatively high concentrations of human serum (1-10%) modified responses to the hormone in a complex manner. As the serum effects varied between samples, it proved impossible to adapt the bioassay by the conventional approach of using a lactogen-depleted serum as a representative matrix. However, as the Nb2 cells were exceptionally sensitive to hGH, the serum effects could be diluted out. We adopted a dilution strategy by which all samples of human serum were included in the bioassay at a concentration of 0.625% or less. A valid assay was obtained, as judged by the criteria of parallelism between diluted samples and hGH standards, and recoveries of spiked samples that were close to 100%. Hormonal specificity was achieved with the use of a highly specific anti-PRL antiserum. A within-assay precision of between 2-5% over the dose range of 0.03-0.96 microgram hGH/L was attained. As only highly diluted samples could be used, the sensitivity of the clinical bioassay was 1.2-2.4 micrograms hGH/L. The between-assay precision was estimated to be 11% and 9% at initial hGH concentrations in serum of 4.8 and 19.2 micrograms hGH/L, respectively. By exploiting the high sample capacity of the eluted stain bioassay system, we followed the changes in bioactivity and immunoactivity of hGH in multiple timed samples after stimulation of hGH secretion in an adult by GHRH. Systematic and progressive changes were observed in the bioactive/immunoactive ratios. Analogous changes were observed after insulin-induced hypoglycemia in a child with short stature. We speculate that the changes in the bioactive/immunoactive ratios reflect alterations in the proportions of the isoforms of hGH in the circulation after acute stimulation.

The effect of a continuous infusion of a somatostatin analogue (octreotide) for two years on growth hormone secretion and height prediction in tall children.

OBJECTIVE: Strategies to limit the final height of tall children have centred on the use of high doses of sex steroids to advance skeletal maturation. This limits therapy to the peripubertal years whereas the greatest gain in height is in the prepubertal years. Prepubertal growth is largely GH dependent and previous work has documented modulation of GH secretion by once or twice daily subcutaneous injection of the somatostatin analogue octreotide. In this study we have determined the effects of a nocturnal infusion of octreotide on height prediction, GH and TSH secretion in tall children. DESIGN: A patient controlled study in which height prediction and 24-hour GH and TSH secretion were compared prior to and during the course of a 2-year treatment programme with a nocturnal infusion of octreotide in a dose of 1-1.5 micrograms/kg body weight given subcutaneously. PATIENTS: Nine tall children (4M; 5F) aged 7-14 years with final height predictions of 179 cm or greater in the girls and between 183 and 202 cm in the boys were studied. MEASUREMENTS: Height prediction using the Tanner-Whitehouse system prior to and at the end of 2 years of treatment. Twenty-four-hour serum GH and TSH concentration profiles, thyroxine, IGF-I and GH responses to GHRH(1-29)NH2 were studied prior to and at the end of the first year of therapy. RESULTS: Treatment with octreotide led to a significant reduction in height prediction in 7 of the 8 children who completed treatment (median reduction 3.5 cm, range +2.8 to -11.5; Wilcoxon, P = 0.05). Twenty-four-hour mean serum GH concentration decreased by 50% (MANOVA, P = 0.03) during therapy and this was accompanied by an increase in the percentage of samples giving values less than 0.5 mU/l (MANOVA, P = 0.02). There was no overall change in the serum GH response to GHRH(1-29)NH2 or serum IGF-I concentrations. Nocturnal serum TSH concentrations were reduced to levels observed during the daytime but these changes had no effect on serum thyroxine values. One patient developed gallstones during the course of therapy. CONCLUSIONS: A nocturnal infusion of octreotide reduces GH secretion and height prediction in tall children. Therapy with somatostatin analogues allows a reduction in growth rate to be instigated in the prepubertal years reducing the actual height from which will commence the pubertal growth spurt.

The effects of oxandrolone on the growth hormone and gonadal axes in boys with constitutional delay of growth and puberty.

OBJECTIVE: We studied the effects of oxandrolone on serum concentrations of LH, FSH, testosterone, GH, SHBG, DHEAS, IGF-I and insulin in boys with constitutional delay of growth and puberty. DESIGN: Ten boys with constitutional delay of growth and puberty, mean age 13.8 years (range 12.4-15.5) were studied. Twenty-four-hour serum concentration profiles of GH, LH and FSH were constructed by drawing blood samples at 20-minute intervals. Three study occasions over a period of 6 months were chosen to assess hormone concentrations before, during and 6 weeks after a 3-month course of oxandrolone (2.5 mg once daily) therapy. RESULTS: Growth velocity increased during oxandrolone treatment and stayed higher after therapy (pre 3.9 +/- 0.5; on 6.3 +/- 0.8; post 6.4 +/- 0.9 cm/year (mean +/- SEM) two way ANOVA, F = 5.3, P = 0.02). Oxandrolone had androgenic effects, suppressing mean serum LH concentrations from 1.7 +/- 0.3 to 1.1 +/- 0.2 U/I and serum testosterone concentrations from 1.9 +/- 0.6 to 0.8 +/- 0.1 nmol/l. SHBG concentrations were also reduced from 130.9 +/- 14.6 to 30.7 +/- 7.3 nmol/l. Serum GH concentration fell slightly from 5.9 +/- 0.6 to 4.8 +/- 0.5 mU/l. After cessation of treatment, there was a significant 'rebound' in mean 24-hour serum LH (2.6 U/l +/- 0.4) and testosterone concentrations (3.2 +/- 0.9 nmol/l) but no change in serum GH concentrations. SHBG values also rose but not to the same extent as those observed before therapy (82.0 +/- 8.4 nmol/l). There were no statistically significant differences in serum concentrations of FSH, DHEAS, IGF-I and insulin over the study period. In a stepwise multiple regression analysis of factors that might influence the growth rate observed, the 24-hour mean serum testosterone concentration and the treatment (on or off) with oxandrolone were the main influences. The relationship was described by the equation Height velocity = 0.69 (24-hour mean serum testosterone concentration)+1.70 (treatment regimen)+3.37 (adjusted R2 = 0.35, F = 8.39, P = 0.001). CONCLUSIONS: Oxandrolone has an androgenic action as shown by changes in serum LH, testosterone and SHBG concentrations and by the lack of effect on FSH. No effect of oxandrolone on the GH axis was documented. We suggest that the growth promoting effects of oxandrolone are related in part to the mild androgenic effects of the steroid and the growth acceleration following oxandrolone withdrawal may reflect increasing total serum testosterone concentrations and decreasing levels of SHBG and progress in puberty.

The relationship between endogenous testosterone and gonadotrophin secretion.

OBJECTIVE: The secretion of luteinizing hormone (LH), follicle stimulating hormone (FSH) and testosterone in the adult male was studied by means of 48-hour profiles. The aim of the study was to examine the effects of the endogenous circadian variation in serum testosterone concentration on LH pulsatility, and to determine the temporal relationships between FSH and LH, and between LH and testosterone by cross-correlation, and the pulse frequency of these hormones by spectral analysis, as revealed by extended sampling periods. DESIGN: Hormone profiles were obtained by 20-minute sampling over 48 hours. SUBJECTS: Six healthy adult males, aged between 21 and 23 years. MEASUREMENTS: LH and FSH were measured using an immunoradiometric assay and testosterone with a solid-phase radioimmunoassay. RESULTS: The profiles showed pulsatile secretion of all three hormones, and a circadian rhythm with levels highest between 0200 and 0600 h. Cross-correlation analysis: There was a significant relationship between LH and FSH (r = 0.5; P = 0.001) at 0 minutes (i.e. no time lag). A pulse of testosterone followed on average 60 minutes after a pulse of LH (r = 0.26; P = 0.001). Fourier transformation: Spectral analysis showed the dominant period for LH and FSH pulsatility to be 200 minutes. The dominant period for testosterone pulsatility was 400 minutes. The effect of endogenous variations in serum testosterone concentrations: The LH profiles were divided into periods when the serum testosterone concentration was high or low, and Fourier transformation carried out on these shorter periods. These transforms showed that the dominant period during high serum testosterone concentration was 180 minutes, and during low serum testosterone was 120 minutes (P < or = 0.025). CONCLUSIONS: FSH and LH are co-secreted, and a pulse of testosterone follows a pulse of LH by 60 minutes. The physiological changes in serum testosterone concentration that occur during the day result in changes in the pulse frequency of LH. Testosterone concentrations thus have rapidly acting feedback activity at hypothalamic level.

Analysis of trough serum growth hormone concentrations: comparison of an immunoradiometric assay and a sensitive ELISA for growth hormone.

OBJECTIVES: We compared a sensitive assay for GH (ELISA) with a conventional immunoradiometric (IRMA) assay with particular reference to the oscillatory activity detected by Fourier transformation and the estimation of trough concentrations using occupancy analysis. DESIGN: Eight healthy adult male volunteers underwent 24-hour profiles during which samples were drawn at 20-minute intervals. Samples were analysed by an ELISA and an IRMA system. MEASUREMENTS: The 24-hour serum GH concentration profiles were subjected to Fourier transformation and to occupancy analysis. RESULTS: No additional GH periodicities could be determined in the ELISA data other than the well documented 180-200-minute periodicity. Median observed concentrations (OC) at 5% occupancy were 0.035 mU/l (range 0.004-0.22) for the ELISA and 0.035 mU/l (range 0.001-0.50) for the IRMA. For all OC parameters, 5, 50 and 95%, there was a good correlation between the ELISA and IRMA systems. The mean difference (bias) between the ELISA and IRMA were -0.05, -0.28 and -1.40 mU/l at OC values of 5, 50 and 95% respectively and the standard deviations of the difference at the same OC values were 0.10, 0.50 and 1.61 mU/l. CONCLUSION: Although there is a qualitative improvement on visual inspection of individual 24-hour serum GH profiles obtained using the ELISA system, there is little additional information gained in terms of pulse periodicity or occupancy analysis.

What is a normal stimulated growth hormone concentration?

In a retrospective analysis, we have compared the response of serum GH concentration to insulin-induced hypoglycaemia in 148 short prepubertal children (114 males, 34 females) aged between 3.9 and 11.9 years with the growth rate of the individual to determine 'cut-off' values for the diagnosis of GH insufficiency. Sixty-three children grew with a height velocity standard deviation score (SDS) greater than -0.8 (group 1), which represents the growth velocity of children progressing along or closely parallel to the third height centile. Eighty-five children had a height velocity SDS of less than -0.8 (group 2). Median peak serum GH concentration responses to insulin-induced hypoglycaemia were 19.9 mU/l (range 1.5-54.4) in group 1 and 9.9 mU/l (range 0.7-46.2) in group 2 (Mann-Whitney; P less than 0.001). Using growth rate as the determinant of normality, the efficiency, sensitivity and specificity of the insulin-induced hypoglycaemia test were calculated using different serum GH concentration cut-off values to diagnose GH insufficiency. In our (Hybritech) assay, a cut-off value of 13.5 mU/l provided optimal performance in terms of efficiency (66%), sensitivity (64%) and specificity (70%). The response of serum GH concentration to insulin-induced hypoglycaemia in short children growing at different growth rates was continuous. Each laboratory measuring serum GH concentrations needs to construct its own 'normal' cut-off value.

Performance of proficiency survey samples in two immunoradiometric assays of human growth hormone and comparison with patients' samples.

The immunoradiometric assay (IRMA) used in our laboratory for the measurement of growth hormone (hGH; somatotropin) performed badly in the national proficiency survey program, the U.K. External Quality Assessment Scheme (EQAS). We compared our assay with another IRMA, which gave similar results for patients' samples and performed adequately in EQAS. The samples from EQAS are collected from patients with polycythemia and fall into two categories: those containing endogenous hGH and those supplemented with pituitary-derived hGH. Analysis of the two groups separately showed that the differences between the two IRMAS were in the measurement of the endogenous hormone. The reason for this appears to be a matrix effect related to the fact that the EQAS serum samples are collected from polycythemic patients.

Rate of change (modulation) of serum growth hormone concentrations is a more important factor in determining growth rate than duration of exposure.

OBJECTIVE: To determine whether duration of exposure to GH and/or rate of change of serum GH concentration are important factors in determining the growth rate of short children. DESIGN: An analysis of parameters of occupancy percentage and rate of change of serum GH concentration was performed as part of a prospective study investigating the relationship between growth and GH in childhood. PATIENTS: Sixty-four short prepubertal children (48 male, 16 female) aged between 4.7 and 11.9 years were studied. Thirty-one children were growing with a height velocity standard deviation score between 0 and -0.8 and were defined as short normal. Thirty-three children were growing with a height velocity standard deviation score less than -0.8 and were defined as short slowly growing. MEASUREMENTS: Twenty-four hour serum GH concentration profiles were constructed by withdrawing samples at 20-minute intervals. Analysis of occupancy percentage was performed on each data array by determining cumulative distributions and plotting these as linear probits against log serum GH concentration. Estimates of peak (OC95), intermediate (OC50) and trough (OC5) occupancies were calculated. A first-order derivative of the concentration-time data array was determined for each profile as a measure of rate changes. RESULTS: First-order derivative values were significantly greater in the short normal group than in the short slowly growing children (short normal median 1.41 mU/l/min; short slowly growing median 0.72 mU/l/min; P less than 0.001). OC95 values were significantly higher in the short normal group (median 19.31 mU/l) than the short slowly growing group (median 7.69 mU/l) (P less than 0.001). There was no difference in OC50 values. OC5 values were lower in short normal children (median 0.20 mU/l) than in the short slowly growing children (0.55 mU/l) (P less than 0.003). The most important factor in determining growth rate was the rate of change in serum GH concentration (FOD). Occupancy percentage played no part in the relationship. The regression equation was Height velocity SDS = 1.16 (In FOD) - 1.03; r = 0.75; P less than 0.001 CONCLUSIONS: These data suggest that the pattern of presentation of GH in the circulation is an important factor in determining target organ response. Although occupancy percentages at differing serum GH concentrations differ between short slowly growing and short normal children, it is the rate of change of the hormone in the circulation which appears to be the more important 'signal' in terms of modulating growth.

A distribution method for analysing the baseline of pulsatile endocrine signals as exemplified by 24-hour growth hormone profiles.

OBJECTIVE: To develop a method for quantifying the distribution of concentrations present in hormone profiles, which would allow an observer-unbiased estimate of the time concentration attribute and to make an assessment of the baseline. DESIGN: The log-transformed concentrations (regardless of their temporal attribute) are sorted and allocated to class intervals. The number of observations in each interval are then determined and expressed as a percentage of the total number of samples drawn in the study period. The data may be displayed as a frequency distribution or as a cumulative distribution. Cumulative distributions may be plotted as sigmoidal ogives or can be transformed into discrete probabilities (linear probits), which are then linear, and amenable to regression analysis. Probability analysis gives estimates of the mean (the value below which 50% of the observed concentrations lie, which we term 'OC50'). 'Baseline' can be defined in terms of percentage occupancy--the 'Observed Concentration for 5%' (which we term 'OC5') which is the threshold at or below which the hormone concentrations are measured 5% of the time. PATIENTS: We report the use of applying this method to 24-hour growth hormone (GH) profiles from 63 children, 26 adults and one giant. RESULTS: We demonstrate that GH effects (growth or gigantism) in these groups are more related to the baseline OC5 concentration than peak concentration (OC5 +/- 95% confidence limits: adults 0.05 +/- 0.04, peak-height-velocity pubertal 0.39 +/- 0.22, giant 8.9 mU/l). CONCLUSIONS: Pulsatile hormone profiles can be analysed using this method in order to assess baseline and other concentration domains.

Reproducibility of 24-hour serum growth hormone profiles in man.

OBJECTIVE: To study the reproducibility of 24-h serum growth hormone (GH) concentration profiles in adults. DESIGN: 24-h serum GH concentrations were constructed by drawing blood samples at 20-min intervals. Four study occasions over a period of 1 year were chosen to assess the reproducibility. SUBJECTS: Six healthy adult male volunteers of normal height and weight and aged between 20 and 22 years. MEASURES: The resulting GH data arrays were analysed by Fourier transformation. Between and within individual variations were calculated and expressed in terms of coefficients of variation and data plotted to show variations between groups and individuals. RESULTS: The frequency component of GH secretion occurred with a dominant periodicity of between 160 and 240 min. Precise estimates of spectral power (strength of oscillatory activity) and period were obtained for group data, but such estimates cannot be inferred from a single profile. There was no significant difference in 24-h mean serum GH concentration over the year of study: occasion 1 (February) mean 2.0 mU/l (SD 0.5); occasion 2 (March) mean 3.7 mU/l (SD 2.8); occasion 3 (July) mean 2.7 mU/l (SD 1.4); occasion 4 (February) 2.5 mU/l (SD 1.5) (mean within individual coefficient of variation 35%, range 9-58). The concentrations of factors known to influence GH synthesis and secretion, insulin-like growth factor I, thyroxine, testosterone and oestradiol, varied little over the year of study. CONCLUSIONS: These data demonstrate that group data are reproducible in terms of oscillatory activity and the amount of GH secreted and that 24-h GH profiles should be used predominantly for analysing group data. The variability between individual profiles limits their value in the investigation of children with growth failure and suspected GH insufficiency.