Comparison of residual C-19 steroids in plasma and prostatic tissue of human, rat and guinea pig after castration: unique importance of extratesticular androgens in men

The concentrations of dehydroepiandrosterone (DHEA), its sulfate (DHEAS), androstenedione (A-dione), testosterone (T) and dihydrotestosterone (DHT) have been measured before and after castration in men and two animal models, namely the rat and the guinea pig. In adult men, the pre-castration levels of plasma DHEAS and DHEA were measured at 1839 +/- 320 and 2.4 +/- 0.5 ng/ml, respectively, while in both animal models, the concentrations of these two steroids were below 0.3 ng/ml. Orchiectomy in men reduced plasma T and DHT levels from 2.9 +/- 0.1 and 0.60 +/- 0.10 to 0.42 +/- 0.21 and 0.05 +/- 0.01 ng/ml (P less than 0.01), respectively, while there was no significant effect observed on DHEAS, DHEA and A-dione levels. By contrast, castration in the rat reduced the low levels of circulating DHEA and A-dione below the detection of the radioimmunoassay (RIA) used. In castrated guinea pig, a small quantity of plasma A-dione (0.07 +/- 0.02 ng/ml) was measured while DHEA was undetectable. Moreover, in the rat and guinea pig, plasma T and DHT levels became undetectable. Following administration of the antiandrogen Flutamide for two weeks in the castrated rat and guinea pig, prostate weight was not further reduced, thus indicating that there is no significant androgenic activity left following castration of these two species. In fact, castration in the rat and guinea pig caused a decrease in prostatic levels of DHT from 4.24 +/- 0.351 and 9.42 +/- 1.43 ng/g, respectively, to undetectable levels. In men, on the other hand, the prostatic DHT levels were only inhibited from 5.24 +/- 0.59 to 2.70 +/- 1.50 ng/g, respectively. As expected, when Flutamide was administered to the rat and the guinea pig, the levels of prostatic steroids remained undetectable while, in men, the DHT content in the prostate was further reduced to undetectable values. In summary, the plasma levels of DHEAS, DHEA, delta 4-dione are markedly different between men and both animal models used and furthermore, measurements of prostatic levels of androgens suggest that the high plasma levels of these steroids are likely responsible for the presence of important amounts of DHT in human prostate after castration.     

Effects of insulin on lipoprotein secretion in rat hepatocyte cultures. The role of the insulin receptor

Insulin inhibits the secretion of lipoprotein components such as triglyceride, phospholipid, and apolipoproteins B and E in primary rat hepatocyte cultures. The aim of this study was to determine whether these hormonal effects are related to the interaction of insulin with its receptor on the surface of cultured hepatocytes. Half-maximal inhibition of secretion of apolipoprotein E and triglyceride occurred at 6 ng/ml porcine insulin, equivalent to a 20% receptor occupancy. When compared to porcine insulin, both guinea pig insulin and desoctapeptide insulin were 60 times less inhibitory on triglyceride and apolipoprotein secretion. These analogs were also 60 times less effective in competing with porcine 125I-insulin for receptor binding. Anti-insulin receptor IgG inhibited binding of porcine insulin to cells in a dose-dependent fashion. However, similar to the hormone itself, it reduced the secretion of triglyceride and apolipoproteins E and B. Preincubation of cells with 200 ng/ml porcine insulin for 15 h caused a 2.5-fold reduction of surface receptor number. These cells were less sensitive to the inhibitory effect of porcine insulin on secretion of triglyceride and apolipoproteins B and E. We conclude that the effects of insulin on lipoprotein processing by hepatocytes in culture are receptor-mediated, can be imitated by antibodies, to the insulin receptor, and are subject to control by receptor down-regulation.     

Neuropeptide Y in guinea pig, rabbit, rat and man. Identical amino acid sequence and oxidation of methionine-17

Neuropeptide Y (NPY) was isolated and characterised from acid-ethanol extracts of rabbit and guinea pig brain. In both instances the chromatographic purification was a two-step procedure of gel filtration followed by reverse-phase high-performance liquid chromatography. The amino acid sequence of rabbit and guinea pig NPY was found to be identical to human and rat NPY as deduced from the cDNA structures. With the exception of the porcine peptide, all mammalian NPYs characterised to date have a methionine residue in position 17. This methionine residue is readily oxidized as indicated by the high degree of spontaneous oxidation of peptides found in the rabbit and guinea pig brain extracts and in NPY extracted from a rat phaeochromocytoma cell line. It is concluded that NPY is among the most highly conserved peptides and that NPYs containing methionine in position 17 are prone to oxidation.     

A highly sensitive enzyme immunoassay of anti-insulin antibodies in guinea pig serum

A highly sensitive enzyme immunoassay of anti-insulin antibodies in guinea pig serum is described. Guinea pig anti-insulin serum was diluted to various extents with nonspecific guinea pig serum and incubated with insulin. After incubation, free insulin was separated from insulin-anti-insulin antibody complex by treatment with dextran-charcoal. Anti-insulin antibodies in the complex were dissociated from insulin by incubation with 0.23 M HCl and inactivated. The amount of dissociated insulin was measured by sandwich enzyme immunoassay using anti-insulin IgG-coated polystyrene balls and affinity-purified anti-insulin Fab'-horseradish peroxidase conjugate. The detection limit of anti-insulin antibodies in guinea pig serum was 6.7 pg/assay or 150 ng/liter of serum. The present enzyme immunoassay was 10,000-fold more sensitive than the previously described enzyme immunoassay, in which insulin-coated polystyrene balls were incubated with diluted guinea pig anti-insulin serum and subsequently with rabbit (anti-guinea pig IgG) Fab'-horseradish peroxidase conjugate.     

Proposed mechanism of insulin-resistant glucose transport in the isolated guinea pig adipocyte. Small intracellular pool of glucose transporters

A marked resistance to the stimulatory action of insulin on glucose metabolism has previously been shown in guinea pig, compared to rat, adipose tissue and isolated adipocytes. The mechanism of insulin resistance in isolated guinea pig adipocytes has, therefore, been examined by measuring 125I-insulin binding, the stimulatory effect of insulin on 3-0-methylglucose transport and on lipogenesis from [3-3H]glucose, the inhibitory effect of insulin on glucagon-stimulated glycerol release, and the translocation of glucose transporters in response to insulin. The translocation of glucose transporters was assessed by measuring the distribution of specific D-glucose-inhibitable [3H]cytochalasin B binding sites among the plasma, and high and low density microsomal membrane fractions prepared by differential centrifugation from basal and insulin-stimulated cells. At a glucose concentration (0.5 mM) where transport is thought to be rate-limiting for metabolism, insulin stimulates lipogenesis from 30 to 80 fmol/cell/90 min in guinea pig cells and from 25 to 380 fmol/cell/90 min in rat cells with half-maximal effects at approximately 100 pM in both cell types. Insulin similarly stimulates 3-O-methylglucose transport from 0.40 to 0.70 fmol/cell/min and from 0.24 to 3.60 fmol/cell/min in guinea pig and rat fat cells, respectively. Nevertheless, guinea pig cells bind more insulin per cell than rat cells, and insulin fully inhibits glucagon-stimulated glycerol release. In addition, the differences between guinea pig and rat cells in the stimulatory effect of insulin on lipogenesis and 3-O-methylglucose transport cannot be explained by the greater cell size of the former compared to the latter (0.18 and 0.09 micrograms of lipid/cell, respectively). However, the number of glucose transporters in the low density microsomal membrane fraction prepared from basal guinea pig cells is markedly reduced compared to that from rat fat cells (12 and 70 pmol/mg of membrane protein, respectively) and the translocation of intracellular glucose transporters to the plasma membrane fraction in response to insulin is correspondingly reduced. These results suggest that guinea pig adipocytes are markedly resistant to the stimulatory action of insulin on glucose transport and that this resistance is the consequence of a relative depletion in the number of intracellular glucose transporters.     

Distribution and characterisation of neuromedin U-like immunoreactivity in rat brain and intestine and in guinea pig intestine

Neuromedin U-8 (NMU-8) is a peptide isolated from porcine spinal cord which contracts blood vessels and the uterus. Antisera were raised against NMU-8 and used in a radioimmunoassay (RIA) together with HPLC to characterize NMU-like immunoreactivity (NMU-LI) in tissues extracts of rat brain and gut and guinea pig gut. Samples of duodenum, ileum and distal colon were taken from both species, and processed for detection of NMU-LI by fluorescence immunohistochemistry. In RIA the antiserum had no cross-reactivity with neuropeptide Y, vasoactive intestinal peptide or the C-terminal hexapeptide of pancreatic polypeptide. Preincubation of antiserum with any of these peptides had no effect on the NMU-LI staining. In rats the highest content of NMU-LI was found in the ileum and the lowest in the cerebral cortex and striatum. HPLC studies showed that at least two molecular forms of NMU-LI were present in both species. In rat small intestine, subpopulations of submucous and myenteric neurones were stained; nerve fibres and terminals within these ganglia and in the mucosa were also seen. NMU-LI was sparse in the muscle. In guinea pig ileum small populations of nerve terminals were seen in both myenteric and submucous ganglionated plexuses. No endocrine cells were stained in either species.     

Increased sensitivity of the enzymatic isotopic assay of histamine: measurement of histamine in plasma and serum.

The use of rat kidney instead of guinea pig brain as the source of histamine-N-methyltransferase for the enzymatic assay of histamine was found to improve the sensitivity of the assay. A partially purified preparation (ammonium sulfate fractionation) of the kidney enzyme was 20- to 50-fold more active than the guinea pig preparation, and sufficient enzyme for 14,000 assays could be prepared from six rats. The kidney enzyme, unlike the guinea pig brain enzyme, was free of interfering enzyme activities and gave low values for assay blanks. The two enzymes otherwise had similar properties. The low blank values permitted direct measurement of histamine in normal plasma without the need to isolate and concentrate histamine from the sample. Plasma histamine levels in normal individuals ranged from 0.2-1.4 (mean 0.6, n = 19) ng/ml.     

Isolation of angiotensin-converting enzyme inhibitor from porcine plasma

An inhibitory peptide of angiotensin-converting enzyme was purified from porcine plasma. The final preparation showed IC50 values of 4.2, 0.6, and 0.9 microgram/ml for angiotensin-converting enzymes from guinea pig serum, rabbit lung, and monkey brain, respectively. The amino acid sequence of the inhibitor was determined as leucyl-valyl-leucine by Edman procedure. This structural observation was supported by mass spectrometric analysis.     

Determination of insulin-like growth factor-I in normal subjects and in patients with growth hormone disorders by radioimmunoassay using biosynthetic homologous peptide

A highly sensitive and specific RIA for IGF-I has been developed using recombinant DNA-derived IGF-I of very high purity and specific antiserum to it. This assay system could detect IGF-I at as low concentrations as 20-30 ng/ml. The intra-assay and interassay coefficients of variation at various concentrations of IGF-I were 4.9 to 6.5% and 5.4 to 8.0%, respectively. The recovery rate of pure IGF-I added to plasma was 77.0 +/- 3.7%. The antiserum did not cross-react with porcine insulin, biosynthetic human insulin, hGH, hEGF, the synthetic C-domain of IGF-I or that of IGF-II, but reacted equally with an analog, Thr59-IGF-I. Plasma IGF-I was extracted by the acid-ethanol method before assay to separate IGF-I from its binding protein. When plasma IGF-I was assayed without extraction, the inhibition curves of serial dilution of plasma samples from several individuals were not parallel to the standard curve of IGF-I. The plasma concentration of IGF-I was 147 +/- 49 ng/ml (mean +/- SD) in 156 normal adults aged from 20-59 years. As reported by others, the IGF-I levels were low in cord plasma (41.8 +/- 23.5 ng/ml) and plasma of patients with GH deficiency (64.6 +/- 42.0 ng/ml), while its levels were high in normal children of pubertal ages (12-13 yr, 365 +/- 126 ng/ml) and in patients with active acromegaly (562 +/- 115 ng/ml). This RIA system is a simple and useful method for determining plasma IGF-I in normal and diseased states.     

Extracts of protozoa contain materials that react specifically in the immunoassay for guinea pig insulin

Extracts of protozoa contain materials that resemble guinea pig insulin, which is noted for its unusual structure and properties. The protozoan derived materials react in the radioimmunoassay for guinea pig insulin; some but not all of these immunoreactive materials migrate on gel filtration in the position of authentic guinea pig insulin. Experiments were done to exclude artifacts in the assay as well as inadvertent contamination by guinea pig insulin. By immunological methods, we segregated the guinea pig type immunoactivity from that which has rat/pork type immunoactivity. These findings extend our studies of extracts of guinea pig tissues which also have these two types of insulin immunoactivities.     

Control of mammary gland fibroblast growth by insulin, growth hormone and prolactin

Fibroblasts isolated from guinea pig mammary glands were cultured in 96 well culture plates in the presence of various concentrations of insulin, growth hormone and prolactin. Insulin (30 micrograms/ml increased uptake of tritiated thymidine by 30%. Higher concentrations of insulin did not result in any further increase in thymidine uptake. Growth hormone alone did not alter thymidine uptake in concentrations of 0 to 250 ng/ml. 300 ng/ml gave thymidine uptake of 136% of controls. In the presence of 20 g/ml insulin, growth hormone (250 ng/ml) increased thymidine uptake to approximately double that of controls. Prolactin alone (300 ng/ml decreased thymidine uptake by 19%. Insulin increased thymidine uptake, but the negative effect of prolactin was still evident above 150 ng/ml.     

Radioimmunoassay of progesterone in peripheral and placental blood of pregnant rabbits and guinea pigs

Radioimmunoassay of progesterone in systemic and placental blood of pregnant rabbits and guinea pigs. 1. The level of progesterone in pregnant rabbits and guinea pigs serum was measured directly (without extraction) using a RadioImmunoAssay (RIA). 2. Hormonal concentrations in systemic blood were shown to increase with gestational age, being at their highest half-way through pregnancy (16.03 +/- 2.63 ng/ml for rabbits; 319.01 +/- 42.10 ng/ml for guinea pigs) and decreasing at the end of the pregnancy. 3. Progesterone was not detectable in rabbit placental blood, whereas a high level of this hormone was found in guinea pig placental blood, which increased with gestational age. From the 28th to the 56th post-coital day, the level increased from 143.22 +/- 13.15 to 283.30 +/- 36.84 ng/ml. 4. The method used enables to measure correctly progesterone concentrations in rabbit and guinea pig serum without extraction.     

Evidence that most of the radioimmunoassayable inhibin secreted by the corpus luteum of the common marmoset monkey is of a non-dimeric form.

Although recent data for several species of primate, including human and marmoset, indicate that the corpus luteum secretes high levels of radioimmunoassayable inhibin, the nature of the immunoreactive (ir) inhibin detected has not been established. In this study, plasma ir-inhibin levels during the ovarian cycle of the marmoset (n = 12 animals) were measured by alpha-subunit-directed inhibin RIA, and values were compared with those estimated by a recently developed two-site immunoradiometric assay (IRMA) specific for inhibin alpha-beta dimer. Consistent with earlier data, plasma levels of ir-inhibin measured by RIA (overall mean value 133 +/- 7 ng/ml; n = 171) reached values 4-fold higher (p less than 0.001) during the luteal phase (222 +/- 20 ng/ml) than during the follicular phase (58 +/- 8 ng/ml), being directly correlated with plasma progesterone levels (r = 0.65; p less than 0.001). In contrast, plasma ir-inhibin levels estimated by IRMA were substantially lower than those measured by RIA (overall mean value 9.62 +/- 1.08 ng/ml; n = 171) and did not vary significantly during the cycle. Administration of a luteolytic dose of cloprostenol during the late luteal phase/early pregnancy led to an abrupt fall in plasma concentrations of progesterone (95%) and alpha-inhibin measured by RIA (82%), whereas dimeric inhibin levels remained unchanged. Analysis of marmoset luteal extracts (n = 5) by RIA, IRMA, and inhibin bioassay yielded inhibin estimates of 102.6 +/- 21.0, 0.632 +/- 0.103, and less than 2.0 ng/mg, respectively, thus confirming that only a very small proportion of the inhibin produced was dimeric (i.e., bioactive) in nature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Radioimmunoassay for insulin-like growth factor I (IGF-I) using biosynthetic IGF-I

We produced antiserum to insulin-like growth factor I (IGF-I), and developed a specific and sensitive radioimmunoassay (RIA) for IGF-I using the biosynthetic IGF-I. This antiserum to IGF-I was specific for IGF-I; no cross-reactivities with multiplication stimulating activity, porcine insulin or human growth hormone (hGH) were detected. The sensitivity was 10-25 pg/tube with 50% displacement at 125 pg/tube. The intra- and inter-assay coefficients of variation for IGF-I were 5.4 and 9.7%, respectively. The plasma IGF-I levels as determined by RIA in normal adults (N = 46), patients with active acromegaly (N = 31), and pituitary dwarfs (N = 31) were 21.6 +/- 1.0, 157.3 +/- 17.0, and 2.5 +/- 0.3 ng/ml (Mean +/- SEM), respectively, indicating the levels were GH-dependent. The plasma IGF-I levels were significantly increased from 2.2 +/- 0.2 to 26.5 +/- 3.2 ng/ml after hGH administrations for three consecutive days in five pituitary dwarfs. The IGF-I levels were low in patients with hypothyroidism and liver cirrhosis, but were normal in patients with chronic renal failure. These data confirm previous reports and this radioimmunoassay proves useful in evaluating plasma IGF-I levels.     

A rapid,sensitive, and easy-to-perform solid phase insulin radioimmunoassay

Accurate measurement of basal insulin release in perifusion, perfusion and low-density β-cell preparations has been difficult with present assays. A simple competitive, equilibrium, 15-hour insulin assay using ¹²⁵I-insulin with microtiter immobilized antubody, has been developed. This method, a Solid-phase-RadioImmunoAssay (SPRIA), is very sensitive and has a broad useful range (1 - 64 μU/ml). For a test series of 4 standard curves, interassay variation between controls of 1, 4, 16, and 64 μU/ml was ±5.2% (SEM) and intra-assay variation over the range of standards between 0.5 to 5.1% (SEM). Nonspecific binding was not significantly different from empty borosilicate culture tubes; 4.0 ± 0.4 and 3.5 ± 0.5 counts/minute (mean ± SEM; n = 54), respectively. This SPRIA can be used with existing γ-counters, while reducing the radioactive and glass waste presently produced by RIA (test-tube can be reused). The radioactive of unused test-tubes was compared againts test-tubes used for greater than 10 assays, values were 3.5 ± 0.5 and 4.4 ± 0.6 counts/minute (mean ± SEM; n = 54), respectively. Results of an oral glucose tolerance test (oGTT) performed on four male Wistar Fursth rats showed a close correlation between SPRIA and RIA insulin values (linear regression, r² = 0.990). This SPRIA measured plasma insulin levels from a human oGTT with a variation of ≤3.7% (SSEM0 between sample triplicates. Standard curves from the three commonly measured insulin isoforms (human, rat and porcine) showed a high correlation (mulitiple linear regression, r² = 0.998, n = 5 standard curves). In order to determine SPRIA's ability to measure acid extracts, insulin recovery from 2N acetic acid was compared against insulin recovery from Dullbecco's Modified Eagles medium (DME). The insulin recovery from 2N acetic acid was greater than 90% of that achieved with DME. in conclusion, an easy-to-perform assay which is deal for the rapid quantification of insulin from isolated islets of Langerhans, isolated β-cells, acetic acid extracts or plasma with greater sensitivity, and less waste than the conventional RIA has been developed.     

Identification of Bradykinin in Mammalian Brain

Abstract: Bradykinin-like activity was purified from acetic acid extracts of saline-perfused rat brains by gel filtration chromatography and two reverse-phase HPLC systems capable of resolving bradykinin from lysyl-bradykinin and other bradykinin analogs and fragments. Addition of [³H]bradykinin to extracts permitted calculation of recoveries and monitoring of chromatographic fractions. Fractions were examined by radioimmunoassay using a potent and highly specific antiserum raised against bradykinin-human albumin conjugates in rabbits. Bradykinin receptor-active material was also measured by radioreceptor assay using guinea pig ileum, as well as by a bioassay with the estrous rat uterus. Active material chromatographed as authentic bradykinin in all systems. Levels of 0.6 pmol/g whole rat brain were detected, with eight times higher levels in the hypothalamus. Activity increased up to 10-fold following treatment with trypsin; treatment with α-chymotrypsin or angiotensin-converting enzyme substantially reduced activity. Similar levels and distribution of bradykinin-like activity were also detected in guinea pig brain extracts. These data substantiate the existence of authentic bradykinin in mammalian brain.     

A rapid radioimmunoassay method of growth hormone in dog plasma.

A rapid and sensitive homologous radioimmunoassay (RIA) system is described for the measurement of growth hormone (GH) in dog plasma. The method requires only 40 hr and is able to detect concentrations of GH as low as 1.0 ng/ml of dog plasma. Antiserum to canine growth hormone (cGH) prepared in monkeys, exhibited a complete cross-reactivity with porcine GH, suggesting that the latter can substitute cGH in a heterologous radioimmunoassay for cGH. Studies on GH regulation performed with this RIA in the unanesthetized dog showed that this species resembles the primate more than the rat or the rabbit.     

Production and secretion of C-19 steroids by rat and guinea pig adrenals

The concentrations of C-19 steroids were measured in guinea pig and rat adrenals before and after castration as well as after stimulation with adrenocorticotropin hormone (ACTH). Characterization of adrenal C-19 steroids was also carried out by isolation with high-performance liquid chromatography and gas chromatography/mass spectrometry (GC/MS). From radioimmunoassay (RIA) data, androstenedione (4-DIONE) and 11 beta hydroxyandrostenedione (11 beta-DIONE) were the major C-19 steroids found in guinea pig adrenals, and castration induced a decrease of 4-DIONE levels only while all other C-19 steroids remained unchanged. In rat adrenals, the major C-19 steroids were 4-DIONE and testosterone, and they were also markedly inhibited after castration. With the exception of 11 beta-DIONE, all other C-19 steroids in circulation were eliminated after castration in both animals species. After ACTH administration in the guinea pig, adrenal 4-DIONE and 11 beta-DIONE levels were markedly stimulated, while an increase of only 11 beta-DIONE was observed in plasma. In the rat, ACTH had a small stimulatory effect on adrenal 52-androstane-3 alpha, 17 beta-diol (3 alpha-DIOL) and plasma 11 beta-DIONE levels. Analysis of guinea pig adrenal steroids by GC/MS confirmed the presence of C-19 steroids in adrenals (namely, 4-DIONE and 11 beta-DIONE) while, in the rat, this could not be confirmed. Our data indicate that production of C-19 steroids occurs in guinea pig adrenals, and 11 beta-DIONE is the major C-19 steroid as well as the only C-19 steroid secreted into the circulation. In the rat, the production of C-19 steroids detected by RIA is not supported by GC/MS data.     

Competitive protein binding assay of progesterone without chromatography

A competitive protein binding assay for the measurement of progesterone in human plasma without chromatographic separation of steroids and recovery evaluation in individual samples is described. It is based on the specificity of the progesterone binding plasma protein (PBP) of the pregnant guinea pig. A dried petroleum ether extract of plasma was incubated with ³H-progesterone and 1600 fold diluted pregnant guinea pig plasma. Bound radioactivity was measured with a dextran coated charcoal suspension technique. Plasma progesterone concentration was obtained by comparison with a standard curve and correction for extraction separately measured for each batch of petroleum ether. The sensitivity was 100 pg. Recovery experiments for progesterone and competing steroids added to plasma respectively showed the accuracy and the specificity of the method. However comparison of the results from assays with and without chromatographic separation of steroids, showed that in the latter-case the specificity was good only for plasmas containing more than 1ng/ml of progesterone. Concentration of progesterone in plasma from men was 0.46±0.14 ng/ml (mean ± S.D) and from post menopausal women 0.30± 0.13 ng/ml.Between days 1 and 13 and days 16 and 22 of the normal menstrualcycle the concentrations were respectively 0.81 ± 0.38 and 12.50 ± 2.96 ng/ml. The variations of the progesterone concentration during pregnancy are also shown.     

Primary Structure and Tissue Distribution of Guinea Pig Gastrin-Releasing Peptide

The primary structure of gastrin-releasing peptide from the guinea pig stomach has been determined by automated Edman degradation and shown to be identical to porcine gastrin-releasing peptide. Extracts of guinea pig brain and small intestine contained both gastrin-releasing peptide and its COOH-terminal decapeptide (neuromedin C) but the stomach extracts contained only gastrin-releasing peptide. Within the small intestine, highest concentrations of gastrin-releasing peptide-like immunoreactivity were found in extracts of the circular and longitudinal smooth muscle layers.