Search

The dodecapeptide angiotensin-(1-12) [Ang-(1-12)] functions as an intracrine/paracrine substrate for local production of angiotensin II. We developed a reliable and specific radioimmunoassay (RIA) method for the measurement of Ang-(1-12) in human plasma and urine using an affinity purified antibody fraction directed towards the C-terminus of the human Ang-(1-12) sequence. The RIA method was applied to quantify the Ang-(1-12) in plasma and urine collected from thirty-four human subjects (29 treated with antihypertensive medicines and 5 untreated patients). Plasma Ang-(1-12) level was significantly higher (P 0.05) in patients with systolic blood pressure ≥140 mm Hg (n = 10) compared to the group with systolic blood pressure140 mm Hg (n = 24). No significant difference (P = 0.22) was found in spot urine between the groups. Our study also shows that the polyclonal antibody neutralizes the cleavage sites of the human Ang-(1-12) from recombinant human chymase (rhChymase) and serum angiotensin converting enzyme (ACE) mediated Ang II generating hydrolysis. Overall, this newly developed RIA method is reliable and applicable to accurately quantify the Ang-(1-12) level in clinical samples (plasma and urine). Further, our in vitro neutralization study suggests that the anti-Ang-(1-12)-antibody might be used as an in vivo therapeutic agent for preventing Ang-(1-12)/Ang II-mediated hypertension and organ damage.

SUMMARY Using a highly specific radioimmunoassay we have measured the concentrations of human growth hormone releasing factor (ir-hGRF) in the peripheral circulation of six individuals with acquired hypothalamic hGRF deficiency. Despite their hypothalamic dysfunction venous plasma ir-hGRF increased normally in every patient after the stimulus of a mixed breakfast, from an average concentration basally of 13.6 ± 6.0pg/ml to a maximum of 29.0 ± 8.4 pg/ml (mean ± SEM) at 120 min. The findings indicate that circulating hGRF is at least in large part extrahypothalamic in origin, which in turn implies a physiological role for hGRF in the periphery.

As an introduction to the Symposium, we have reviewed the early steps in glucagon research from its discovery in 1923 to the establishment of the basics of the physiology and pathophysiology of the hormone after the description of a sensitive and specific radioimmunoassay by Unger and his co-workers in 1959.

Area under the concentration-time curve (AUC) has been advocated as a better parameter to monitor cyclosporine A than trough concentrations. Up to now, more than 100 equations to estimate AUC using a limited sampling strategy have been published, but not all have been validated.Eight equations for AUC0-12h and two for AUC0-8h were validated. Concentrations of cyclosporine A were analyzed by high-performance liquid chromatography (HPLC) and a specific radioimmunoassay (RIA) method. Forty male renal transplant patients were included in the study. Blood samples were taken predose and at 0.5, 1, 1.5, 2, 3, 5, 8, and 12 hours after the morning dose when the patient was in steady state. The percentage prediction error (%pe) was used for an assessment of the performance of the equations. Mean %pe less than ± 15% and absolute %pe less than 30% in 95% of predictions were considered to be acceptable. Other possibilities such as %pe less than 25%, 20%, and 15% were also tested.Eight equations for AUC0-12h met the requirements using both assays, six in the HPLC set only and four in the RIA set only. The highest precision was obtained with AUC0-12h = 123.792 + 1.165*C1h + 3.021*C3h + 7.33*C8h proposed by de Mattos et al. The mean %pe was 1% ± 8% (-16 to 19) for HPLC (values given as mean ± standard deviation [range]) and -1 ± 5 (-17 to 10) for RIA. Mean absolute %pe was 7 ± 5 (0.0 to 19) for HPLC and 4 ± 4 (0.0 to 17) for RIA. For clinical use, the most suitable equation was AUC0-12h = 363.078 + 8.77*C1h + 3.07*C3h proposed by Wacke et al, which produced the second lowest %pe and used two sampling points in the period of 1 to 3 hours after dose. The mean %pe was -7 ± 10 (-25 to 25) for HPLC and 2.3 ± 6 (-10 to 17) for RIA. Mean absolute %pe was 10 ± 7 (0.4 to 25) for HPLC and 5 ± 4 (0.0 to 17) for RIA. The equation: AUC0-8h = 55.37 + 2.89*C0h + 1.08*C1h0.9*C2h + 2.23*C3h proposed by Foradori et al met the criteria with 95% of prediction with absolute %pe less than 15% in the HPLC set and 10% in the RIA set.The validation of equations is of major importance for prediction precision, whereas the analytical method for limited sampling strategy proposals had no influence. Because of the wide interassay variability, it is also important to know which analytical method was used for AUC calculation when interpreting the results.

Salusin-alpha was recently shown to exert anti-atherosclerotic effects and its potential role as a clinical marker for atherosclerosis has been proposed. We determined serum salusin-alpha concentrations in 99 patients across a diverse range of renal functions and urinary salusin-alpha excretions in 12 patients with non-dialyzed renal failure using a highly sensitive and specific radioimmunoassay. Serum salusin-alpha concentrations in patients with moderate to advanced renal insufficiency (eGFR30 ml/min/1.73 m(2)) were significantly lower than those with preserved renal function (eGFR60 ml/min/1.73 m(2)) (6.1 + or - 2.4 pmol/l vs. 11.8 + or - 1.1 pmol/l, p0.05). Since renal failure is frequently associated with atherosclerosis, we analyzed the relationship between serum salusin-alpha and eGFR after excluding patients with advanced atherosclerotic diseases. The serum salusin-alpha level was correlated with eGFR values (n = 94, p0.005). Patients with renal insufficiency showed reduced urinary salusin-alpha excretion, but the magnitude of the reduction was less than that for the decrease in serum salusin-alpha. Consequently, their salusin-alpha clearance often exceeded endogenous creatinine clearance levels. In conclusion, the decreased serum concentrations of salusin-alpha, an anti-atherosclerotic peptide, may be associated with impaired renal function, suggesting a potential role of decreased salusin-alpha in the acceleration of atherosclerosis in chronic kidney diseases. Urinary salusin-alpha may originate from the renal tubules, and may not necessarily represent the peptides filtered at the glomerulus.

We compared a midregion (44-68) human parathyroid hormone (hPTH) specific radioimmunoassay (M-RIA) with a C-terminal (65-84) hPTH specific radioimmunoassay (C-RIA). The M-RIA discriminated 21 of 23 patients with primary hyperparathyroidism from 95 normals (normal range: 22.4-106.5 pmol/l). With the C-RIA 12 patients including the 2 patients not discriminated by the M-RIA had immunoreactive PTH (iPTH) values within the normal range of this assay (normal range: undetectable to 88.6 pmol/l). To investigate the reasons for these different abilities of separation, hyperparathyroid sera were subjected to gel-filtration and analyzed using the two assays. Intact PTH appeared to have a major influence on the immunoreactivity of circulating PTH in the M-RIA. In contrast, the C-RIA showed the highest immunoreactivity with midregion-C-terminal PTH fragments. Hyperparathyroid sera not discriminated by the C-RIA but with elevated iPTH in the M-RIA showed decreased amounts of midregion-C-terminal PTH fragments, while intact PTH comprised the highest amount of total circulating PTH immunoreactivity in the M-RIA. From the present results we conclude that the superiority of the M-RIA is due to the determination of intact PTH which is preferable for clinical measurements with relation to the diagnosis of primary hyperparathyroidism.

Immunoreactivity of synthetic human beta-endorphin analogs with various chain lengths has been examined using a specific radioimmunoassay. It was found that beta-endorphin-(1--21) and analogs of shortened chain exhibit no immunoreactivity, whereas beta-endorphin-(1--15) possesses significant in vitro opiate activity. It appears that immunoreactivity of beta-endorphin resides in the COOH-terminal segment of residues (22--31). The data also show the lack of correlation between opiate and immunological activities of beta-endorphin.

Summary. Investigation of the family of a patient presenting with haematuria revealed seven cases in three generations showing a prolonged thrombin clotting time. A dysfibrinogenaemia was confirmed when purified fibrinogen from an affected member of the family was shown to also exhibit a prolonged thrombin clotting time. No molecular abnormality could be demonstrated using electrophor-etic and immunological techniques. However, using a specific radioimmunoassay to fibrinopeptide A a major defect has been localized to a delay in the rate of release of this peptide by thrombin when the abnormal fibrinogen is converted to fibrin. This case of dysfibrinogenaemia has been tentatively designated fibrinogen Manchester.

Blood concentrations of thyrotropin-releasing hormone (TRH, Protirelin) were measured by a specific radioimmunoassay in 16 euthyroid subjects after intravenous (200 micrograms), nasal (2 mg) or oral (40 mg) administration of TRH. TRH blood levels peaked 2 min after i.v. administration (13,400 +/- 1,020 fmol/ml), 10 min after nasal (5,000 +/- 1,800 fmol/ml) and 150 min after oral (2,650 +/- 1,080 fmol/ml) administration. The degradation of TRH followed an exponential curve, giving a half-life of 6.5 min. The disappearance rate gave a "half-life" of 22 min in the nasal application group and of 31 min in the oral group. Maximal concentration of stimulated thyrotropin-stimulating hormone (TSH) always occurred 30 min after the peak TRH concentration without any correlation with the TRH concentration time integrals, suggesting that TSH secretion is dependent on a continually rising TRH blood level.

Galanin, a neuropeptide, has important effects on hormone secretion from the hypothalamus and pituitary, and may also be involved in important biological processes such as pain, memory, and food intake. Yet, there is limited knowledge about how these processes are reflected by circulating galanin. To study the levels and molecular forms of galanin in the human circulation, plasma was analysed from 27 healthy subjects, 14 women and 13 men, using two extraction methods and a specific radioimmunoassay for human galanin. After extraction on Sep Pak C-18 columns, plasma galanin-like immunoreactivity (galanin-LI) in the healthy men was 6.3 +/- 2.5 pmol/l (mean +/- SD, n = 12), which was higher than in the women, 4.1 +/- 1.5 pmol/l (n = 14, p = 0.010). A small increase in galanin-LI was seen with age in the women (r = 0.54, p < 0.05) but there was no significant difference between pre- and postmenopausal women. Galanin immunoreactivity after Sep Pak and immunoextraction correlated (r = 0.74, p < 0.001) the levels being higher after immunoextraction (p < 0.001). Gel chromatography disclosed heterogeneity of circulating galanin-LI with the majority eluting as homologs with a molecular weight higher than synthetic human galanin. Homologs smaller than galanin were also found. Sep Pak C-18 extraction eliminated the majority of the higher molecular forms. In conclusion, circulating galanin-LI was found to be higher in men and to be present mainly as molecular forms larger than synthetic galanin.

The purpose of this study was to validate an equine-specific osteocalcin (OC) radioimmunoassay (RIA) for use in donkeys and to establish age-related changes in serum OC concentrations in healthy donkeys. Serial dilutions of donkey serum showed parallelism with standard curves obtained with the equine-specific OC RIA. There was a tight linear regression between donkey serum OC values obtained with the equine specific OC RIA and a commercially available bovine-specific OC RIA. Serum OC levels of 27 healthy donkeys, analysed with the equine-specific OC RIA, showed a tight negative logarithmic regression with age. Least square means and standard error of serum OC values were 67 +/- 10 microg/l in 0.6-4-year-old donkeys (group 1), 32 +/- 9 microg/l in 5-9-year-old donkeys (group 2) and 15 +/- 11 microg/l in > 9-year-old donkeys (group 3). Serum OC values were significantly (p < 0.01) higher in group 1 than in groups 2 and 3. The equine-specific OC RIA may be a useful and practical tool to assess bone metabolism and skeletal diseases in donkeys.

Using newly developed ghrelin specific radioimmunoassay (RIA) combined with high performance liquid chromatography (HPLC), expression and release of ghrelin/des-n-octanoyl ghrelin in mouse testicular Sertoli TM4 cells were demonstrated. The expression and release of the peptides were significantly higher than those of Leydig TM3 cells under the condition used. Testosterone (1—100 ng/mL) increased dose-dependently ghrelin release from Leydig TM3 cells but not from Sertoli TM4 cells. Both TM3 and TM4 cells possessed growth hormone secretagogue receptor (GHS-R). Implication of Sertoli cells in ghrelin expression in the testis has never been discussed. Apart from species-specificity or/and age-dependency in expression of ghrelin in different cell types of the testis, the expression and release of the peptide in TM4 cells which were found in this study raise an issue of physiological significance of ghrelin in Sertoli cells of the testis.

The study was conducted to develop a sensitive and specific radioimmunoassay (RIA) for the measurement of pepsinogen in porcine serum, and to use this test for the determination of pepsinogen concentrations in serum samples from fetuses and pigs of different ages. Compared to a previously described RIA, major improvements were made concerning the use of specific polyclonal antibodies and the use of an appropriate buffer. The assay was able to detect pepsinogen concentrations of/=0.2 ng/mL. The recovery of pepsinogen was close to 95%. The intra-assay coefficients of variations ranged between 3.9 and 7.5% whereas the interassay ranged between 8.8 and 11.9%. These percentages correspond to a satisfactory accuracy and reproducibility of the assay. No cross-reactions were observed with the main commercially available products of the aspartic proteases family except porcine pepsin cross-reacted over 62.5 microg/mL. Pepsinogen concentrations increased steadily with increasing age of the fetuses and the pigs (P0.05). Pepsinogen concentrations (+/-SE) in fetuses of 90-100 (n=24) and 100-110 days of pregnancy (n=36) were 0.5+/-0.1 and 5.3+/-1.3 ng/mL, respectively. In pigs of 21, 98, and 213 days of age, the pepsinogen concentrations were 290.6+/-10.8, 343.1+/-17.9 and 383.5+/-15.3 ng/mL, respectively. The results demonstrate that RIA is accurate and can be used easily to assess pepsinogen concentrations in pig sera. The test may constitute a valuable tool in epidemiological surveys and in studies related to gastric diseases in pigs.

Glycation of insulin, resulting in impaired bioactivity, has been shown within pancreatic beta cells. We have used a novel and specific radioimmunoassay to detect glycated insulin in plasma of Type 2 diabetic subjects.Blood samples were collected from 102 Type 2 diabetic patients in three main categories: those with good glycaemic control with a HbA(1c) less than 7%, moderate glycaemic control (HbA(1c) 7-9%) and poor glycaemic control (HBA(1c) greater than 9%). We used 75 age- and sex-matched non-diabetic subjects as controls. Samples were analysed for HbA(1c), glucose and plasma concentrations of glycated insulin and insulin.Glycated insulin was readily detected in control and Type 2 diabetic subjects. The mean circulating concentration of glycated insulin in control subjects was 12.6+/-0.9 pmol/l ( n=75). Glycated insulin in the good, moderate and poorly controlled diabetic groups was increased 2.4-fold ( p0.001, n=44), 2.2-fold ( p0.001, n=41) and 1.1-fold ( n=17) corresponding to 29.8+/-5.4, 27.3+/-5.7 and 13.5+/-2.9 pmol/l, respectively.Glycated insulin circulates at noticeably increased concentrations in Type 2 diabetic subjects.

This study describes for the first time the development and validation of a sensitive and specific radioimmunoassay (RIA) for equine osteocalcin (OC) quantification using purified equine OC as standard, tracer, and immunogen for antibody formation in rabbits. The assay allowed to measure equine serum OC levels with a sensitivity of 0.2 ng/mL. Immunoreactive serum OC values of clinically normal, different-aged horses ranged from 3.68 to 127.31 ng/mL. Intra- and inter-assay coefficients of variation (CV) were 6.2 and 8.2%, respectively. Serial equine serum sample dilutions were linear. The recovery of equine OC from equine serum samples ranged from 93.88 to 107.9%. There was a tight correlation between OC values measured with the equine-specific OC RIA and two commercially available bovine-specific OC RIA kits. However, highest serum OC values were obtained with the equine-specific OC RIA. In conclusion, our equine-specific OC RIA is sensitive, linear, accurate, precise, and reproducible. The assay allowed to quantify OC in equine serum samples and might, therefore, be used to monitor equine osteoblast activity associated with bone diseases, exercise, therapy forms or diet.

The purpose of the study was to investigate the prevalence of gastric lesions and to provide diagnostic values for serum pepsinogen in non-infected pigs and in pigs with gastric disease. In an abattoir survey, the pepsinogen concentrations were measured in the serum from 62 non-infected pigs, 33 pigs with gastric lesions and 17 pigs infected with Hyostrongylus rubidus, using a specific radioimmunoassay (RIA). The mean (±SE) pepsinogen concentrations in the serum of non-infected pigs, in pigs with gastric ulcers, and in pigs with a heavy H. rubidus infection were 630.8±39.2 ng/ml, 1084.5±166.2 ng/ml and 1095.2±102.3 ng/ml, respectively (p

We described a new, specific and easy to use radioimmunoassay (RIA) of estrone sulfate (E1S) in males. After synthesis of an E1S-6-(O-carboxymethyl) oxime hapten then coupling to BSA, we obtained a specific anti-E1S antiserum. Although the cross-reactivity of DHEAS with our anti-E1S antiserum was low (CR=0.002%), we confirmed the absolute necessity of separating plasma DHEAS from plasma E1S, before E1S RIA, because in plasma, DHEAS is present at levels 3–6000-fold higher than E1S, which generally is ignored. Thus, we elicited an easy separation of DHEAS from E1S, by a fast chromatography on in-house minicolumns. This new RIA, was applied to the determination of E1S plasma normal values in males. In 27 young men ( 1.97 nmol/l ±1.07 nmol/l and in 63 untreated healthy aged men (>55 years), 1.80 nmol/l ±1.21 nmol/l . No significant difference was seen between young and older subjects. The ranges of E1S plasma levels in these subjects were rather large and the ratios between the highest and the lowest E1S plasma levels were seven in the young group and 23.4 in the older group. No decrease of E1S plasma levels was observed with ages. Contrary to large interindividual E1S plasma level variations, the intraindividual variations have been found to be no significant. Correlations between E1S and unconjugated estrogens, E2 and E1 were 0.22 (P=0.016) and 0.51 (0.001), respectively.

Asphyxia and reperfusion induced changes in the plasma and cerebrospinal fluid (CSF) concentrations of alpha-melanocyte-stimulating hormone (alpha-MSH) were studied in newborn pigs using a specific radioimmunoassay technique. Cardiovascular and metabolic failure induced by neonatal asphyxia resulted in a 3-fold, significant (P0.05) increase in plasma alpha-MSH levels, whereas the hormone concentration in CSF was significantly (P0.05) reduced by 50% during postasphyxial reperfusion. Our data indicate an asphyxia-induced release of alpha-MSH, and suggest a discordant regulation of plasma and CSF concentrations in newborn pigs.

A single antibody, equilibrium, species specific radioimmunoassay (RIA) for catfish insulin was developed and validated for measurements of plasma levels of insulin in juvenile and adult channel catfish. The RIA appeared to be sensitive (20 pg/tube) and stable with intra-assay coefficient of variation between 5.7% and 3.1% and inter-assay coefficient of variation of 6.5%. The blood was collected from two strains of catfish, USDA 103 and Norris, which differ in food consumption and growth rates, the former strain consuming more food and growing faster (Silverstein et al. 1999). The experiments conducted from December to May included fasting and feeding periods from 5 to 14 days as well as either intravenous or intraperitoneal administration of glucagon-like peptide known for its anorexic effect in both mammals and fish (Silverstein et al. 2001). As in other piscine species studied before, fed fish had higher plasma insulin content than food-deprived fish. Feeding status prevailed over other treatments as the primary physiological factor determining circulating plasma insulin levels.

International audience; Recombinant chicken leptin was used to produce an antiserum in order to develop a specific and sensitive radioimmunoassay (RIA) for chicken leptin in plasma and serum. We have used either murine or chicken leptin as tracer and competition curves were performed using recombinant chicken leptin. Variations in leptin plasma levels in different chicken strains and various nutritional states were correlated with the physiological status. Leptin plasma concentrations were regulated by the nutritional state with higher levels in the fed state as compared to the fasted state (3.36 +/- 0. 13 versus 2.78 +/- 0.11 ng/ml) and being dependent upon the age. Higher leptin levels were found in 22 week-old as compared to 15 week-old layer chickens (2.709 +/- 0.172 versus 1.478 +/- 0.102 ng/ml). We have also shown that the multispecies leptin RIA kit (LINCO Inc.) underestimated leptinemia compared to the chicken leptin- specific RIA reported here. In conclusion the RIA developed in the present study is specific to the chicken and thus may be considered as powerful tool for investigating the physiological significance of leptin in chickens.

An improved neurosecretory granule (NSG) preparation of the shore crab, Carcinus maenas sinus glands, enriched for members of the crustacean hyperglyc?mie hormone family (CHH) and almost devoid of contaminating subcellular fractions, was obtained by differential and density gradient centrifugations. CHH was determined in an aliquot of each fraction by means of a specific radioimmunoassay, and purity of the fractions by means of electron microscopy of pellet sections from duplicate tubes. Fraction F3 from the first discontinuous gradient centrifugation was enriched for CHH-containing NSG and purified on a second, shallower discontinuous gradient, and fraction F3b proved to contain solely CHH granules as revealed by EM and RIA criteria.

A sensitive and specific radioimmunoassay for orexin A was developed. Orexin A immunoreactivity was found to be confined to the central nervous system (CNS) with the highest concentrations in the hypothalamus, inferior and superior colliculi and brainstem. Within the hypothalamus, the highest levels were found in the lateral and posterior hypothalamus. These regions had a greater orexin A content in females compared to males. The orexin A content of hypothalamic regions did not change with fasting and no difference was noted in hypothalami of rats fed a high fat diet. The hypothalamic orexin A content was not different in obese Zucker rats compared to lean controls. Thus, orexin A has a wide distribution in the CNS, but appetite regulation may not be its main function.

Die Anwendbarkeit der Bestimmung von 17-Hydroxyprogesteron (17-OHP) im Speichel zur Kontrolle der Behandlung des angeborenen adrenogenitalen Syndroms (AGS) infolge C21-Hydroxylase-Defektes wurde untersucht. 17-OHP im Speichel wurde mit Hilfe eines spezifischen Radioimmunoassay nach Extraktion mit Dichlormethan bestimmt. Die Menge von extrahiertem Speichel fur den Assay war mit 100 µl gering. Zwischen den Bestimmungen von Speichel- und Serum-17-OHP-Konzentrationen, die an gepaarten Speichel- und Serumproben (11 angeborene Nebennierenhyperplasien und 6 normale Kontrollpersonen) durchgefuhrt wurden, zeigte sich eine gute Korrelation (r = 0,93; P

Our observation that dispersed cultures of neurohypophysis obtained from adult rats are capable of synthesizing and releasing oxytocin and vasopressin is unexpected, because in whole animals these hormones are known only to be stored, not to be produced in the posterior lobe of the pituitary. The hormone content of cell culture medium was elevated from 0 to 129 +/- 14 pg/mg protein for oxytocin and from 0 to 42 +/- 4 pg/mg protein for vasopressin during two weeks as determined by specific radioimmunoassay. By molecular mass and structure determination (tandem mass spectrometry) we have proved that the supernatant of the cell cultures contains not only immunologically but mass spectrometrically identified neurohypophyseal hormones.

Melatonin was measured using a specific radioimmunoassay in 1 strain of outbred mice (OF1 Swiss) and 4 strains of inbred mice, 2 of them being known to synthesize melatonin (CBA and C3H) and the 2 others being controversial (BALB/c and C57BL/6). In this study, the 5 mouse strains were able to synthesize melatonin, but the basal levels as well as the diurnal variations were very different from one strain to another. CBA and C3H strains showed a clear-cut day-night rhythm of pineal melatonin concentration, with peak levels of 276 ± 22 pg/pineal in CBA and 135 ± 12 pg/pineal in C3H. In BALB/c, the authors confirmed the presence of a very short melatonin peak (15 min) in the middle of the dark period. In C57BL/6 and OF1 Swiss, a very small but significant peak was observed in the middle of the darkness. In the former, another small peak was also observed at light onset. Whether these very small peaks, which may be related to the deficience of N-acetyl transferase activity reported by others, have a physiological meaning remains to be determined.

Some alterations in hypothalamo-pituitary-thyroid axis occur during aging. In this study we evaluated the changes induced by aging in pituitary and thyroid iodothyronine-deiodinase (DI) activities, and in serum T4, T3 and TSH. Groups of 6–18 female Dutch-Miranda rats aged 3–5 months (young adults) were studied in parallel with similar groups of old (10–12 months) and senescent (24–30 months) animals. DI activities were determined in the microsomal fraction of pooled pituitary or thyroid glands (6 glands per pool), using T4 as substrate and DTT as cofactor; the T3 formed was measured by specific radioimmunoassay. Serum T3, T4 and TSH were measured by specific radioimmunoassays. Serum T4 was significantly decreased in both groups of aged rats, but serum TSH was unaffected. Serum T3 was just slightly decreased in the senescent rats. Total pituitary DI activity was significantly decreased in the aged rats (10–12 and 24–30 months). Both type I and type II DI activities were affected, although the decrease in type I DI only became significant in the senescent rats. In contrast to its effect in the pituitary, aging does not decrease, even slightly, the DI activity in the thyroid gland. The thyroid DI activity may contribute to the unaltered serum T3 levels found in aged rats in the present study.

A specific antiserum for morphine-6-glucuronide (M6G) has been raised in a rabbit in response to immunization with a novel hapten: Protein conjugate (N-aminobutylnormorphine-6-glucuronide-thyroglobulin). Cross-reactivity with morphine and structurally related compounds was found to be negligible as expected from the nature of this immunogen. Using this antiserum, a simple, rapid and robust radioimmunoassay (RIA) has been developed for determination of M6G in samples of human plasma. The assay has a sensitivity of 0·05 ng/mL using 100 μL sample volumes and affords complete recovery of M6G over the range 2–200 ng/mL. The presence of morphine or morphine-3-glucuronide at concentrations up to 100 times the levels of M6G did not result in any measurable interference. Close agreement was obtained between M6G results obtained using the RIA and a specific high-performance liquid chromatography assay. This RIA offers an attractive alternative to existing methods for the determination of M6G in human plasma and will facilitate further metabolic and pharmacokinetic studies of morphine and M6G in the clinical setting.

Histidyl-proline diketopiperazine (CHP) is a cyclic dipeptide that is found in many animal tissues, most notably brain and gut. It has been found to have a variety of biologic actions and has been postulated to play a role in appetitive behavior and energy metabolism. This study was conducted in order to characterize the secretory pattern of CHP during a 24 h fast. Four subjects (2 obese and 2 lean) were studied during the latter 24 h of a 36 h fast. Blood was sampled every 10–15 min and assayed for CHP concentration using a specific radioimmunoassay. Analysis revealed that circulating CHP oscillates in humans and that diurnal variation occurred but only in the obese subjects.

C-type natriuretic peptide (CNP) is assuming increasing importance in cardiovascular disease, and in adults its plasma levels are related to clinical and functional disease severity. Data are scarce regarding the reference values for CNP in infancy. Aim of this study was to assess the reference intervals for CNP in human healthy newborns and infants. Plasma CNP was measured in 121 healthy children divided into: 41 newborns (age 0-3 days), 24 newborns (4-30 days), 22 infants (1-12 months) and 32 children (1-12 years). A group of 32 healthy adult subjects (age 64 ± 1 years) was also studied. CNP was measured by a specific radioimmunoassay. Between- and within-assay variability resulted ≤ 30 and 20%, respectively and analytical sensitivity 0.77 ± 0.05 pg/tube. Plasma CNP resulted significantly higher in children than in adult subjects (13.6 ± 1.2 pg/ml vs. 7.4 ± 1.0 pg/ml, p=0.030). When the results were analyzed as a function of the age the reference intervals for plasma CNP resulted: 11.6 ± 2.1 pg/ml for newborns (0-3 days), 16.4 ± 3.7 pg/ml for newborns (4-30 days), 15.4 ± 2.7 pg/ml for infants (1-12 months), 13.6 ± 2.3 pg/ml for children (1-12 years) [p=0.01 newborns (4-30 days) vs. adults; p=0.03 infants (1-12 months) vs. adults]. CNP showed the highest concentrations after 12h of life with a peak between 4 and 5 days of life and with a progressive decline afterwards. According to these data at least five different reference intervals for CNP determinations should be used. These observations may be helpful for future clinical application of CNP in human children.

*Measured in whole blood by specific radioimmunoassay or high performance liquid chromatography. The actual results depend on the laboratory in which the measurement is made. In this series we have outlined the uses of measuring the plasma concentrations of some drugs and given guidelines on how such measurements should be made and interpreted. The box summarises the target plasma concentrations for each of the drugs. In each case there is a concentration below which a therapeutic effect is unlikely and a concentration above which the risk of toxicity is high. These two concentrations imply a therapeutic range for each drug, but remember that there are circumstances in which strict adherence to a

We investigated the relationship between plasma insulin-like growth factor I (IGF-I) levels and the body mass index (BMI) in 558 healthy adults (238 males and 320 females), aged 21-80 yrs. The subjects were divided into three groups based on the BMI: 1) underweight group (BMI20, n = 145), 2) normal group (BMI 20-25, n = 179) and 3) overweight group (BMI25, n = 234). Blood samples were obtained after overnight fasting and plasma IGF-I concentrations were measured by specific radioimmunoassay after acid-ethanol extraction. Plasma IGF-I levels declined with age in each group and the regression lines were parallel. In both sexes of each decade, mean plasma IGF-I values were lower in the underweight and overweight groups than in the normal group. Age- and sex-matched analysis revealed that plasma IGF-I values were positively correlated with BMI under 25, whereas plasma IGF-I was reversely correlated with BMI beyond 27. These findings suggest that the plasma IGF-I levels are influenced by body composition independently of aging in adults, aged 21-80 yrs.

Osteocalcin is the most abundant non-collagenous protein produced in the process of bone formation. A specific radioimmunoassay has been developed using a rabbit antiserum raised against osteocalcin extracted from rat bone. The sensitivity of the assay was tested in male and female rats under different experimental conditions: ovariectomy led to a mild increase in circulating osteocalcin (70.6 +/- 6.9 vs 51.6 +/- 6.3 ng/ml; p < 0.05) and deprivation of dietary calcium elevated plasma levels further (119 +/- 6.3 ng/ml; p < 0.01). As expected, pharmacological enhancement of bone turnover with calcitriol produced a significant increase in plasma osteocalcin (296 +/- 24.1 vs 89.5 +/- 5.1 ng/ml; p < 0.01), whereas prednisolone, a steroidal compound known to inhibit osteoid mineralization, significantly reduced circulating concentrations of this protein (70 +/- 7.4 vs 100 +/- 6.3 ng/ml; p < 0.05). Plasma kinetics recorded in female rats between birth and the 100th week revealed a highly significant (p < 0.001) elevation peaking at the third week (231 +/- 70.6 ng/ml) and slowly declining to reach values measured at birth (41.3 +/- 9.2 ng/ml) at the 16th week (47 +/- 4.6 ng/ml). Subsequently, a small but significant (p < 0.05) decline towards senescence was recorded. The osteocalcin surge preceded the period of rapid growth (weeks 3 to 11) estimated by vertebral length progression, showing a tendency to stabilize as growth spurt slowed down. A moderate but significant (p < 0.01) increment was observed after mating (87.8 +/- 5.1 vs 69.5 +/- 4.0 ng/ml). Although plasma osteocalcin remained stable during lactation, average levels were elevated in comparison with age-matched non-pregnant controls.(ABSTRACT TRUNCATED AT 250 WORDS)

A radioimmunoassay for the measurement of rat pancreatic polypeptide (RPP) in serum or plasma has been developed and characterized using a new guinea-pig anti-rat-PP antibody. The assay provides a high degree of sensitivity and lacks cross-reactivity (CR less than 0.01%) to neuropeptide Y and peptide YY. It also does not interact with PPs of other species or peptide hormones namely, amylin, glucagon, human insulin, human-PP, human-proinsulin, rat C-peptide and rat insulin. The assay employs synthetic rat PP as standards from concentrations of 21-2100 pg/ml (i.e., 5-500 pM) and produces a sensitivity limit of 19 pg/ml (4.5 pM) PP at +/- 3 S.D. The intra- and interassay % coefficient of variations are 6.4% and 5.9%, respectively. The % recovery of RPP added to rat serum samples ranges from 98% to 103%. Assay of serum volumes ranging from 25 microliters to 100 microliters does not significantly alter the expected RPP level. The migration patterns of rat serum PP and that of a synthetic RPP are identical by Sephadex G-50 chromatographic analysis. The mean values of fasting and a 2 h post-feeding plasma RPP levels in normal rats are 40 +/- 2 and 80 +/- 10 pg/ml (9.5 pM and 19.0 pM), respectively. Rat-PP release during insulin induced hypoglycemia in conscious rats rises from 38 +/- 5 pg/ml to 261 +/- 34 pg/ml (9.0 to 62.1 pM, P less than 0.005) by 30 min. Additionally, the antibody used in this study cross-reacts well with mouse-PP as determined by linear serum dilution curves, thus making it useful in the measurement of murine-PP. In conclusion, we have developed and validated a sensitive and specific rat-PP assay. This assay provides a new tool for the reliable measurement of PP in physiologic studies using rat and mouse animal models.

The role of vasoactive intestinal peptide (V.I.P.) in nerve regeneration was investigated by assessing the changes in immunoreactive V.I.P. levels in rat sciatic nerves following injury and repair. 60 rats were divided into three surgical groups and one control group: In group I (primary repair), sciatic nerves were divided and immediately repaired; in group II (secondary repair), sciatic nerves were divided and repaired two weeks later; in group III (no repair), sciatic nerves were divided and not repaired; and in group IV (controls), sciatic nerves were exposed but not divided. Animals were sacrificed at three days and at weekly intervals. Their sciatic nerves were extracted and assayed for V.I.P. concentrations by a specific radioimmunoassay. The mean V.I.P. concentration varied between 22 and 46 pg./mg. protein in the control nerves and between 60 and 529 pg./mg. protein in all other groups. In the three surgical groups the levels were significantly higher in proximal than in distal stumps. Following nerve injury, there was an increase in V.I.P. concentration in the injured and repaired areas. This increase was greater in injured non-repaired areas and was highest in the first 48 hours, but continued during regeneration. The accumulation of V.I.P. in divided nerves occurred in response to nerve injury.

Sensitive and specific radioimmunoassay (RIA) methods for the analysis of morphine and hydromorphone in human plasma samples using commercially available materials were developed. The limit of quantitation was 0.3 ng/mL of plasma for morphine and 50 pg/mL of plasma for hydromorphone. An extraction step preceding the RIA quantitatively removed morphine, leaving 99% morphine-3-glucuronide (M3G) and 95% hydromorphone-3-glucuronide (H3G) in the aqueous phase. The specificity of the morphine RIA method for the analysis of clinical samples was confirmed by HPLC quantitation. The antiserum used in the hydromorphone RIA method cross reacted slightly with H3G at 0.66%. However, analysis of clinical samples using the direct versus the extraction RIA showed that the extraction step was necessary for the specific determination of hydromorphone in pharmacokinetic studies. After extraction, only 0.033% of the H3G present in plasma samples would be observed as interference for the free hydromorphone. The RIA methods were shown to be accurate and reproducible with almost 100% recovery of morphine and hydromorphone. They offer convenient alternatives to chromatographic methods for pharmacokinetic studies.

Cyclosporine in whole blood samples from renal and cardiac transplant recipients was measured by high-performance liquid chromatography (HPLC) and by CYCLO-Trac SP specific radioimmunoassay (RIA). The fresh samples assayed by RIA were remeasured in a second laboratory after storage at -20 degrees C for two to six months and good interlaboratory agreement was obtained on the 59 samples assayed (y = 0.926x + 14.8 micrograms/L, r = 0.982). The calibration curve for RIA was not influenced by fresh whole blood, hemolyzed whole blood or serum matrix from normal volunteers. However, comparison of the RIA results from patient samples with those from an HPLC procedure showed that RIA values averaged about double those measured by HPLC. The difference is attributable to differences between the blood matrix of transplant and nontransplant subjects, rather than exclusively to the apparent nonspecific nature of the antibody.

The distribution and frequency of bombesin immunoreactive neuroendocrine (NE) cells including neuroepithelial bodies (NEB) was analyzed morphometrically in lung sections from 25 infants who died of sudden infant death syndrome (SIDS) and 25 control infants. The control group included infants age-matched to those with SIDS, as well as subjects ranging in age from early to late infancy, to define the postnatal development of pulmonary NE-cell system. Quantitative analysis was performed on lung sections immunostained with monoclonal antibody against bombesin and the contents of bombesin-like peptide in lung extracts were measured by a specific radioimmunoassay (RIA). In control infants, the frequency of NE cells was high at birth but decreased dramatically during the first year of life. In SIDS infants, the frequency of NE cells, the size of NEB, and the mean concentration of bombesin-like peptide detected by RIA were significantly increased compared to those values for age-matched controls. These findings suggest hyperplasia of bombesin-immunoreactive NE-cell system in the lungs of SIDS infants. Since NEB are thought to function as hypoxia-sensitive airway chemoreceptors and since these cells are prominent in the neonates but decline postnatally, we speculate that chronic hypoxia and/or developmental delay may be responsible for this alteration in the lungs of SIDS victims. Potential dysfunction of pulmonary NE-cell system, compounded by other abnormalities in the autonomic regulation of respiration may be of importance in the pathogenesis of SIDS.

Diurnal patterns of growth hormone (GH) in peripheral plasma were compared between mature German Landrace (GL) boars (n = 6), GL sows (n = 4) and European wild boars (n = 5). Blood samples were drawn at 20-min intervals through indwelling jugular vein catheters for 24 h. Some of the animals were sampled repeatedly, so twelve 24-h profiles were available for GL boars, six for GL sows and seven for wild boars. The GH concentrations were determined by a specific radioimmunoassay. GL boars revealed significantly higher (P ≤ 0.001) GH concentrations than GL sows (boars: 5.06 ± 2.34 ng ml−1; sows: 3.70 ± 1.01 ng ml−1). In GL boars a clear diurnal pattern with distinct GH pulses during daytime and a less pulsatile GH release during the night was found, whereas in females no diurnal pattern was obvious. Mean GH levels in wild boars (1.97 ± 1.03 ng ml−1) were significantly lower (P < 0.001) than in both groups of GL pigs. The diurnal pattern of GH pulses in wild boars, however, coincided with the pattern of GH release in GL boars (r = 0.26; n = 73; P ≤ 0.05).

Digoxinlike materials (DLMs) are compounds found in animals that (1) cross-react with digoxin antibodies in specific radioimmunoassay procedures and (2) bind to Na+, K(+)-ATPase and inhibit its activity. DLMs may play a part in the regulation of electrolyte composition. We have determined DLM concentrations in breast cyst fluids and found that these fluids were a rich source of DLMs. Total DLMs were elevated in fluids with type 1 electrolyte composition (high K+ and low Na+ content) when compared to fluids with type 2 composition (low K+ and high Na+ content). Chromatographic studies showed that the basis for the increase was the presence of an additional DLM in the type 1 cyst fluids rather than an increased concentration of the same DLM present in both types of fluids. We propose that this additional DLM may have a role in the accumulation of K+ ions in type 1 fluids. In addition, 7 of 80 cyst fluid samples subjected to high-performance liquid chromatography had a large UV-absorbing peak at 6.4 minutes. Four of these samples were taken from women who later developed breast cancer; information on the subsequent medical history of the other 3 women was unavailable. We suggest that the presence of this material in cyst fluid may be a marker of risk for development of breast cancer.

A specific radioimmunoassay method was developed for H-Met(O2)-Glu-His-Phe-D-Lys-Phe-NH(CH2)8-NH2(ebiratide)., an analogue of adrenocorticotropic hormone. The sensitivity was 30pg/ml. Recovery of ebiratide from plasma was almost 100%, and the intra and inter-assay coefficients of variation were less than 15%. Plasma levels and urinary excretion of ebiratide were determined in six healthy male volunteers after intravenous administration of ebiratide (150μg). Ebiratide was eliminated from the plasma in a biphasic pattern, with half-lives of 3.2min (first phase) and 3h (second phase), and 0. 76% of the dose was excreted in urine. These results indicate that this radioimmunoassay method is suitable for analyzing the pharmacokinetics of ebiratide.

16alpha-Hydroxy-dehydroepiandrosterone (16alpha-OH-DHEA) belongs to the products of extensive DHEA metabolism in mammalian tissues. It is a precursor of 16alpha-hydroxylated estrogens, increased levels of which are associated with autoimmune disorders. A highly specific radioimmunoassay of unconjugated 16alpha-OH-DHEA was developed and evaluated. Polyclonal rabbit antisera were raised against 3beta,16alpha-dihydroxy-17,19-dione-19-O-(carboxymethyloxime) and 3beta,16alpha-dihydroxy-7,17-dione-7-O-(carboxymethyloxime) BSA conjugates. Two methods were used for preparation of the conjugates. Homologous radioiodinated derivatives with tyrosine methyl ester were prepared as tracers. While antisera to 7-CMO cross-reacted with DHEA as much as by 58%, the cross-reaction of the chosen antiserum prepared via 19-oxogroup by micellar conjugation technique with 16beta-OH-DHEA was only 0.13% and with all other structurally related steroids, including DHEA were lower than 0.01%. The detection limit was 0.017 pmol (5.7 pg)/tube, the average intra- and inter-assay coefficients of variation were 8.2 and 11.4%, respectively. Mean recovery of serum spiked with 16alpha-OH-DHEA varied between 80 and 110%, the results were independent on sample dilution. 16alpha-OH-DHEA concentrations in 18 randomly selected sera, including 6 samples from patients with thyroid cancer were compared with results obtained by earlier GC-MS method. Physiological levels of 16alpha-OH-DHEA in 316 sera (184 females and 132 males) analyzed so far varied between 0.0 and 1.86 nmol/l.

The neuropeptide galanin has important effects on hormone secretion from the hypothalamus and pituitary, and it may also be involved in central biological processes such as pain, memory, and food intake. Yet, there is limited knowledge about how these processes are reflected by circulating galanin. To study the levels and molecular forms of galanin in the human circulation, plasma was analysed from 26 healthy subjects, 14 women and 12 men, using two extraction methods and a specific radioimmunoassay for human galanin. Galanin-LI levels in unextracted plasma were higher (141-191 pmol/L) than after immunoextraction (3.4-30.7 pmol/L) and Sep Pak extraction (2.2-12.6 pmol/L). Galanin immunoreactivity after Sep Pak and immunoextraction correlated (r = 0.74, p0.001). Galanin-LI levels were significantly higher in the men than in the women (p = 0.01) after Sep Pak extraction. A small increase in galanin-LI was seen with age in the women (r = 0.54, p0.05). The proportion of Sep Pak extracted galanin-LI increased with age in the women (r = 0.73, p0.05)) but not in the men.