Immunofunctional assay of human growth hormone (hGH) in serum: a possible consensus for quantitative hGH measurement.

Confirmation of the diagnosis of GH deficiency in adults and children involves provocative testing for human (h) GH. Different commercially available immunoassays yield largely discrepant results in the measurement of GH levels in human serum. These discrepancies result in doubtful relevance of cut-off levels proposed for GH provocative testing. We have developed an immunofunctional assay method that allows quantitation of only those GH forms in circulation that possess both binding sites of the hormone for its receptor and thus can initiate a biological signal in target cells. An anti-hGH monoclonal antibody recognizing binding site 2 of hGH is immobilized and used to capture hGH from the serum sample. Biotin-labeled recombinant GH-binding protein in a second incubation step forms a complex with those hGH molecular isoforms that have both binding sites for the receptor. The signal is detected after a short third incubation step with labeled streptavidin. The assay is sensitive (detection range, 0.1-100 micrograms/L) and has average inter- and intraassay precisions of 10.3% and 7.3% respectively. Endogenous GH-binding protein does not interfere with the hGH result; placental lactogen slows no detectable cross-reaction in this immunofunctional assay. The degree of immunofunctionally active hGH forms in serum samples, calculated by comparison of immunofunctional assay and RIA results, varied between 52-93%. We propose this immunofunctional assay for GH measurement as a new reference method for hGH quantitation in serum. The immunofunction assay translates only hGH forms into an assay signal that are capable of dimerizing GH receptors and, thus, of initiating a biological effect in target cells.

Effects of a two-week physiological dehydroepiandrosterone substitution on cognitive performance and well-being in healthy elderly women and men.

The levels of dehydroepiandrosterone (DHEA) and its sulfate ester DHEAS decrease with age after a peak around 25 yr. Animal studies as well as the first studies in humans have generated the idea that DHEA replacement in elderly subjects may have beneficial effects on well-being and cognitive functions. In the present experiment 40 healthy elderly men and women (mean age, 69 yr) participated in a double blind, placebo-controlled DHEA substitution study. For 2 weeks subjects took 50 mg DHEA daily, followed by a 2-week wash-out period and a 2-week placebo period. The treatment sequence was randomized in a cross-over design. After 2 weeks of DHEA or placebo, psychological and physical well-being as well as cognitive performance were assessed using several questionnaires and neuropsychological tests. All subjects had low DHEAS baseline levels. DHEA substitution lead to a 5-fold increase in DHEAS levels in women (from 0.67 +/- 0.1 to 4.1 +/- 0.4 micrograms/mL; P < 0.001) and men (from 0.85 +/- 0.1 to 4.5 +/- 0.4 micrograms/mL; P < 0.001). DHEA, androstenedione, and testosterone levels also increased significantly in both sexes (all P < 0.001). No significant changes were observed in insulin-like growth factor I or insulin-like growth factor-binding protein-3 levels. DHEA replacement had no strong beneficial effect on any of the measured psychological or cognitive parameters. Only women tended to report an increase in well-being (P = 0.11) and mood (P = 0.10), as assessed with questionnaires. They also showed better performance in one of six cognitive tests (picture memory) after DHEA. However, after Bonferroni alpha adjustment, this difference was no longer significant. No such trend was observed in men (P > 0.20). Likewise, no beneficial effects of DHEA substitution could be observed in any of the other tests of the neuropsychological test battery in either sex (all P > 0.20). In conclusion, the present data do not support the idea of strong beneficial effects of a physiological DHEA substitution on well-being or cognitive performance in healthy elderly individuals.

Monoclonal anti-acid-labile subunit oligopeptide antibodies and their use in a two-site immunoassay for ALS measurement in humans.

Quantification of the acid-labile subunit (ALS) has to date been restricted to immunoassays utilizing polyclonal antibodies. By immunization with N-terminal and C-terminal specific ALS oligopeptides, we generated monoclonal antibodies (mAbs) that target ALS-specific sequences outside the nonspecific leucine-rich repeats in the ALS molecule. For mAb selection, a special screening method was developed. Monoclonal antibody 5C9, which targets the N-terminus of ALS, is immobilized and the anti-ALS mAb 7H3, directed against the C-terminus, is biotinylated and used as tracer Ab. Due to the extreme pH-lability of ALS, changes in immunorecognition of ALS were investigated after acidification for protein unfolding in different pH ranges and in a time-dependent manner. It was determined that acidification of the serum samples to pH 2.7 for 30 min, followed by neutralization and dilution to 1:100 was the optimal acid-neutralization method. For standardization purposes, a serum pool derived from healthy volunteers was assigned the value 1 U/ml ALS. The sandwich assay has a working range with a linear dose-response curve in a log/log system between 0.005 and 10 U/ml. ALS levels in seven acromegalic patients ranged from 2.0 to 4.2 U/ml, and in 12 untreated growth hormone deficient patients from 0.036 to 0.986 U/ml (mean=0.45 U/ml). After 12 months of growth hormone therapy, ALS levels increased significantly to 1.18+/-0.45 U/ml (mean+/-SD; p<0.0006). The increase ranged from 0.48 to 1.4 U/ml. The change in ALS with growth hormone (GH) therapy correlated closer with the change in IGF-I (r=0.798, p=0.0057; Spearman rank correlation) than with the change in insulin-like growth factor binding protein (IGFBP3; r=0.549, p=0.057). This specific sandwich assay for the measurement of ALS provides a potentially valuable indicator of growth hormone secretory status. With this mAb-based immunofluorometric assay, the nonspecific detection of other proteins containing leucine-rich repeat sequences can be excluded.

Interactions of insulin-like growth factor (IGF)-II and growth hormone in vivo: circulating levels of IGF-I and IGF-binding proteins in transgenic mice.

To study interactions between insulin-like growth factor-II (IGF-II) and growth hormone (GH) in vivo, we crossed hemizygous transgenic mice carrying phosphoenolpyruvate carboxykinase (PEPCK)-IGF-II fusion genes with hemizygous PEPCK-bovine GH (bGH) transgenic mice. Offspring harbouring both transgenes (IB), the IGF-II transgene (I) or the bGH transgene (B), and non-transgenic littermates (C) were obtained. Blood samples were taken before (end of week 12) and after (end of week 14) the mice had received a diet high in protein and low in carbohydrates to stimulate PEPCK promoter-controlled transgene expression. Mean serum GH concentrations of both B and IB mice corresponded to 900 ng/ml and increased more than twofold (P < 0.001) after 1 week of the high-protein diet. GH concentrations in controls and I mice were less than 20 ng/ml. Serum IGF-II concentrations in I and IB mice were three-to fourfold higher than those in C and B mice. Whereas IGF-II concentrations were not changed by the high-protein diet in the last two groups, serum IGF-II increased significantly in I (P < 0.001) and IB mice (P < 0.05). This increase was significantly (P < 0.05) less pronounced in IB than in C and I mice. Circulating IGF-I concentrations were about twofold (P < 0.001) higher in B and IB than in C and I mice, and showed a tendency to be lower in I than in C and in IB than in B mice when animals were maintained on the standard diet. The high-protein diet did not change circulating IGF-I concentrations in controls and B mice, but resulted in a significant reduction of serum IGF-I concentrations in I (P < 0.05) and IB mice (P < 0.001). Consequently, after PEPCK-IGF-II transgene expression was stimulated, serum IGF-I concentrations were significantly (P < 0.05) lower in I than in C and in IB than in B mice. Serum IGF-binding protein (IGFBP)-2 concentrations were significantly (P < 0.05) higher in I mice than in all other groups when mice were maintained on the standard diet, with a tendency to reduced IGFBP-2 concentrations in B mice. After the high-protein diet, serum IGFBP-2 concentrations did not change in C and I mice, but increased by two- to threefold in B and IB mice (P < 0.001). Serum IGFBP-3 concentrations tended to be greater in B and IB than in C and I mice, but these differences were mostly not significant. IGFBP-4 concentrations were significantly (P < 0.001) increased by GH overproduction in B and IB mice. Our data suggest that the reduction in circulating IGF-I concentrations by increased IGF-II is most probably due to the limited serum IGF binding capacity and the short half-life of free IGFs, rather than to a reduction in GH-dependent IGF-I production. Effects of GH overproduction on serum IGFBP-2 concentrations depend on dietary factors and may be both inhibitory and stimulatory.

Synthesis of a cortisol-biotin conjugate and evaluation as a tracer in an immunoassay for salivary cortisol measurement.

Cortisol 3-(o-carboxymethyl)oxime (C3-CMO) and a commercially available biotin-hydrazide derivative were used to synthesize a C3-CMO-biotin conjugate. C3-CMO was converted into a N-hydroxysuccinimide ester derivative which in a second reaction step was allowed to interact with the hydrazide derivative of biotin. This simple-to-perform synthesis yielded a conjugate suitable for use as a tracer in immunoassays for cortisol measurement. Employing biotin as the primary probe in a competitive solid phase immunoassay allows for variable end point determination by means of commercially available labeled avidin or streptavidin derivatives. Streptavidin-Europium was used in conjunction with the DELFIA-system for time-resolved fluorometric end point measurement (TR-FIA) throughout the study. In addition, colorimetric end point determination (ELISA) using streptavidin-alkaline phosphatase as a secondary probe was established and evaluated. Both forms of this non-isotopic assay showed excellent correlation with a commercially available radioimmunoassay adapted for salivary cortisol measurement. The lower detection limit was 0.43 nM for a 50 microliters salivary sample. The intra-assay coefficient of variation was 6.7, 4.7 and 4.0% at cortisol concentrations of 2.2, 5.5 and 13.2 nM, respectively (n = 37), and the corresponding inter-assay coefficients of variation were 9.0, 8.6 and 7.1% (n = 50). The competitive immunoassay requires 1.5 h incubation time and shows robust and reproducible performance. The C3-CMO-biotin conjugate allows for sensitive and flexible end point determination of salivary cortisol levels in immunoassays.

A time-resolved fluoroimmunoassay for cortisol in unextracted bovine plasma or serum with optimized procedures to eliminate steroid binding protein interference and to minimize non-specific streptavidin-europium binding.

A time-resolved fluoroimmunoassay (TR-FIA) for human salivary cortisol was adapted for the measurement of cortisol in unextracted bovine blood plasma and serum. It has been demonstrated that the binding of cortisol binding plasma proteins (CBPP) to the cortisol-biotin primary probe cannot be eliminated by means of cortisol releasing agents. Complete inactivation of CBPP was achieved by heating water diluted samples for 30 min at 80 degrees C. The high non-specific binding (NSB) of the streptavidin-europium secondary probe, encountered during preliminary experiments, was shown to be caused by an interaction with bovine serum albumin (BSA) and could be reduced partly by the addition of heparin. It was also shown that the ability to bind streptavidin-europium nonspecifically is not a general property of proteins since bovine gamma-globulin and gelatin lack this behaviour. The advantage of a highly reduced NSB, resulting from the use of a BSA free assay buffer, is not limited to this particular assay but is also beneficial for other procedures based on specific measurement of streptavidin-europium fluorescence. The detection limit for a 20 microl sample was 0.5 ng/ml. The intra-assay coefficients of variation for control samples with cortisol concentrations of 71.1, 39.2 and 10.3 ng/ml were 8.2, 7.9 and 11.3% (n = 16). The corresponding inter-assay coefficients of variation were 7.3, 9.0 and 11.2% (n = 73). Correlation with a commercially available radioimmunoassay, preceded by diethylether extraction of the sample, was 0.97 (n = 88).

A non-isotopic immunoassay for guanosine 3':5'-cyclic monophosphate using a cyclic GMP-biotin conjugate as tracer.

2'-O-monosuccinylguanosine 3':5'-cyclic monophosphate was coupled to N-biotinyl-1,8-diamino-3,6-dioxaoctane after converting succinyl-cGMP into its N-hydroxysuccinimide active ester. Isolation and purification of the succinyl-cGMP-biotin conjugate was performed with FPLC using reversed phase chromatography. The synthesis described yielded a conjugate suitable for use as tracer in immunoassays for the cGMP measurement in plasma and urine samples. Employing biotin as the primary probe in a competitive solid phase immunoassay allows for flexible end point determination by means of commercially available labeled streptavidin derivatives. Streptavidin-europium was used in conjunction with the DELFIA-system for time-resolved fluorometric end point measurement (TR-FIA), streptavidin-horseradish peroxidase was used for colorimetric end point determination (EIA). Both non radioactive immunoassay systems showed excellent correlation with the reference radioimmunoassay, good sensitivity and reproducibility. The succinyl-cGMP-biotin conjugate was shown to be stable for more than two years without any apparent loss of chemical stability or immunological reactivity.

Reduced natriuresis during weightlessness.

The kidney response to weightlessness was measured in one volunteer during a 1-week space mission. Shortly after entering microgravity and later during the mission, consecutive urine sampling periods were monitored, covering in total about 50% of the inflight time. Preflight references were a sequence of ground-based experiments, which evaluated body fluid metabolism with different degrees of standardization. Additional variables, such as circadian rhythms and cortisol-associated stress, were also monitored. In contrast to current hypotheses, the volunteer showed a pronounced reduction in natriuresis and diuresis during the entire space flight, despite a considerable weight loss. For the first time, the urinary excretion of the renal natriuretic peptide urodilatin was also measured. Both, during the preflight experiments and during weightlessness, close correlations between urodilatin excretion and sodium excretion were observed. However, the correlation between natriuresis and urodilatin excretion was considerably altered during weightlessness. We conclude that the loss of body weight during space flight is not related to an increased renal fluid loss and that urodilatin might counteract the decrease in renal excretion observed in weightlessness.

A time-resolved fluorescence immunoassay for the measurement of testosterone in saliva: monitoring of testosterone replacement therapy with testosterone buciclate.

Monitoring of testosterone replacement therapy requires a reliable method for testosterone measurement. Determination of salivary testosterone, which reflects the hormone's biologically active plasma fraction, is a superior technique for this purpose. The aim of the present study was to establish a new sensitive time-resolved fluorescence immunoassay for the accurate measurement of testosterone levels in saliva and to validate it by monitoring testosterone replacement therapy in eight hypogonadal men. A clinical phase I-study with the new ester testosterone buciclate was performed to search for new testosterone preparations to produce constant serum levels in the therapy of male hypogonadism. After two control examinations eight male patients with primary hypogonadism were randomly assigned to two treatment groups (n = 2 x 4) and given single doses of either 200 mg (group I) or 600 mg (group II) testosterone buciclate intramuscularly. Saliva and blood samples were obtained 1, 2, 3, 5 and 7 days post injection and then weekly for three months. The time-resolved fluorescence immunoassay for salivary testosterone shows a detection limit of 16 pmol/l, an intra-assay CV of 8.9% (at a testosterone concentration of 302 pmol/l), an inter-assay CV of 8.7% (at a testosterone concentration of 305 pmol/l) and a good correlation with an established radioimmunoassay of r = 0.89. The sample volume required by this method is only 180 microliters for extraction and duplicate determination. The assay procedure requires no more than three hours. In group I (200 mg) testosterone did not increase to normal levels either in saliva or in serum. However, in group II, androgen levels increased significantly and were maintained in the normal range for up to 12 weeks with maximal salivary testosterone levels of 303 +/- 18 pmol/l (mean +/- SE) and maximal testosterone levels of 13.1 +/- 0.9 nmol/l (mean +/- SE) in serum in study week 6 and 7. The time-resolved fluorescence immunoassay for salivary testosterone provides a useful tool for monitoring androgen status in men and women and is well suited for the follow-up of testosterone replacement therapy on an outpatient basis. The long-acting ester testosterone buciclate is a promising agent for substitution therapy of male hypogonadism and in combination with testosterone monitoring in saliva offers an interesting new perspective for male contraception.

Development of a highly sensitive nonisotopic immunoassay for the determination of salivary 17-hydroxyprogesterone: reference ranges throughout childhood and adolescence.

A sensitive nonisotopic immunoassay for the determination of 17-hydroxyprogesterone (17-OHP) levels in saliva was developed. The new time-resolved fluorometric immunoassay employs a specific polyclonal anti-17-OHP antiserum immobilized onto microtiter plates, a 17-OHP-biotin conjugate as a tracer, and streptavidin-europium a as secondary probe. The lower detection limit of the assay is 23.6 pmol/L (mean -3 s of a 22-fold zero determination) corresponding to 0.39 pg/well. The coefficients of intraassay variation are 8.8, 5.3, and 8.3% at the respective concentrations of 90.9, 454.5, and 1363.5 pmol/L. The coefficients of interassay variation are 8.8, 5.3, and 8.3% at the respective concentrations. Saliva was collected in commercially available devices. Reference ranges were established using 394 saliva samples from 132 healthy children, adolescents, and adults. Morning, midday, and evening levels of 17-OHP levels in saliva varied significantly in all age groups with morning levels being higher than midday and evening levels. Saliva samples (n = 57) were also obtained from 18 children with congenital adrenal hyperplasia (CAH). Salivary 17-OHP levels in the limited number of CAH patients studied ranged from 121 to 106,050 pmol/L. In conclusion 1) a new, sensitive nonisotopic immunoassay for measurement of 17-OHP in saliva has been developed; 2) reference ranges for healthy children, adolescents, and adults have been established; 3) there is a circadian pattern of 17-OHP levels in saliva at all ages; and 4) measurement of 17-OHP in saliva should be further evaluated over a longer period of time as a potentially reliable and powerful technique to monitor metabolic control in patients with CAH. As 17-OHP levels in saliva are stable for > 10 wk at 4 degrees C, the technique is ideally suited for outpatient sampling.

Salivary cortisol levels throughout childhood and adolescence: relation with age, pubertal stage, and weight.

The measurement of cortisol in saliva has become a reliable tool for both the scientist and the clinician for studying adrenal cortical function in the adult. We have measured salivary cortisol in samples from 138 healthy infants, children, and adolescents, and from 14 adults. Saliva samples were obtained at home using a cotton swab and a saliva-collecting tube at 800, 1300, and 1800 h before meals. Cortisol was measured using a time-resolved fluorescent immunoassay. Cortisol levels in saliva ranged from less than 2 nmol/L up to more than 100 nmol/L. Cortisol levels were age-dependent. Interestingly, after the age of 6 y, cortisol levels correlated significantly with pubertal stages (analysis of variance). No sex difference was found. In addition, cortisol morning levels and daily cortisol levels (area under the curve from three measurements) increased with body weight and body mass index. The highest cortisol levels were measured in saliva of children younger than 1 y. No circadian variation was evident before the age of 9 mo. After 1 y of age, salivary cortisol levels varied in a circadian fashion. The measurement of salivary cortisol levels is an attractive way of testing adrenal function in infants and children. It provides a reliable tool for the determination of the physiology and developmental characteristics of cortisol metabolism.