Measurement of serum prolactin and the effect of ketamine anesthesia on serum prolactin levels in cynomolgus monkeys (Macaca fascicularis)

The effect of an anesthetic, ketamine, on the serum prolactin level was examined in wild-originating female cynomolgus monkeys (Macaca fascicularis) imported from South East Asia. Serum prolactin levels were measured by the homologous radioimmunoassay system which was developed for human prolactin. The validity was confirmed by using an extract of pituitary gland from a female cynomolgus monkey as well as serum and amniotic fluid from a pregnant monkey. Additionally, serum luteinizing hormone (LH) levels were determined by the radioreceptor assay system developed in our laboratory using Leydig cells collected from rat's testes as a receptor fraction. The experiment was repeated three times at one-month interval, using twenty animals that were divided into three groups consisting of 5, 7 and 8 monkeys each. In the first experiment, the first group was injected with physiological saline and the second and third groups were intramuscularly given ketamine at a dose level of 5 mg/kg B.W. and 15 mg/kg B.W., respectively. In the second experiment, the first and second groups were given ketamine at a dose of 5 mg/kg B.W. and of 15 mg/kg B.W., respectively, and the third group was served as control injected with saline. In the third experiment, the first and third groups were administered with 15 mg/kg and 5 mg/kg of ketamine and the second group was injected with saline. In short, all of the twenty monkeys received the three different treatments for two months. The serum prolactin level showed a marked increase after the administration of ketamine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Species specificity of radioreceptor assay and radioimmunoassay for rat FSH

Species specificity of the radioreceptor assay (RRA) for rat FSH, in which pregnant mare serum gonadotropin (PMSG)-treated immature rat ovary was employed as the receptor, was compared with that of NIAMDD rat FSH radioimmunoassay (RIA). In the RIA system, pituitary preparations from mammals only showed significant crossreaction. Their inhibition curves, however, were not always parallel to the standard curve. On the other hand, in the RRA system, the pituitary preparations from mammals, avians, lizard and amphibians competitively inhibited the binding of radioactive rat FSH to the ovarian receptor. Only the pituitary preparation from dog salmon failed to show any crossreaction in the RRA system. These results indicated that this RRA system would be useful for the measurement of FSH or gonadotropins of the pituitaries from mammals to amphibians.     

Purification and characterization of luteinizing hormone from the dromedary (Camelus dromedarius)

Luteinizing hormone (LH) has been purified from 150 dromedary pituitaries and its partial physicochemical, biological and immunological characterization has been achieved. Purification of the hormone was monitored by a porcine LH radioreceptor assay (RRA). In this system, the final camLH preparation exhibited an activity 0.6-fold that of highly purified porcine LH. The acid half-dissociation of camLH at equilibrium was observed at pH 4.2. A homologous camLH RRA was developed using the testicular plasma membrane fraction from prepubertal camels and radioiodinated, highly-purified camLH. Pituitary and chorionic gonadotropins (CG) from several mammalian species were compared to camLH in this system. The equine gonadotropins eLH and eCG were shown to be 6 times less potent in the camel RRA than in the porcine RRA, whereas the LH from other species exhibited similar activities in both systems. This particularity of camel LH receptors offers a new tool for the study of structural features of gonatropin interactions with their receptors.     

Luteinizing hormone receptors in ovaries of the cynomolgus monkey (Macaca fascicularis) during the menstrual cycle

Ovarian luteinizing hormone (LH) receptors were characterized using ovarian tissues from 17 cynomolgus monkeys at different phases of the menstrual cycle. Low binding affinity receptors for ¹²⁵I-LH were observed throughout the menstrual cycle. The binding affinity of these receptors for LH (< 12 × 10¹⁰ M^?1) was approximately the same as that of ovarian LH receptors previously reported in human and nonhuman primates. In addition, high-affinity receptors (17?85 × 10¹⁰ M^?1) were also detected at the mid-luteal phase, during which a large functional corpus luteum was present. Thus the high-affinity LH receptors appear with the formation of the corpus luteum and disappear with its regression. Almost no fluctuation of binding capacity was observed throughout the menstrual cycle (32?112 fmol/ mg of ovarian tissue). The high-affinity LH receptor was judged to be derived from the functional corpus luteum.     

A functional radioreceptor assay of alpha-V-beta-3 (alphavbeta3) inhibitors in plasma: application as an ex vivo pharmacodynamic model

Development of alphavbeta3-integrin inhibitors has been hampered by a lack of pharmacodynamic endpoints to identify doses that inhibit alphavbeta3 in vivo. To address this need, we developed an alphavbeta3 radioreceptor assay (RRA) that could be performed in 100% plasma. The RRA was based on 125I-echistatin binding to plate-immobilized alphavbeta3. Small molecule alphavbeta3 inhibitors efficiently competed echistatin binding to alphavbeta3 when the assay was carried out in buffer. However, when carried out in 100% plasma, the RRA revealed a 45 to >3000-fold loss in compound potencies. The losses in potency reflected, in part, the high plasma protein binding by the compounds examined. The RRA was adapted as an ex vivo pharmacodynamic model. Echistatin binding was measured in the presence of plasma harvested at timed intervals from rats dosed with select compounds. Using this pharmacodynamic model, compound and dose selection was optimized for further testing in models of corneal angiogenesis. Moderate anti-angiogenic activity was achieved when rats were dosed sufficient to achieve sustained (>50%) plasma inhibition through the trough interval. Thus, the RRA provided a simple technique to rank order compound potency in plasma, and could find general use as an ex vivo pharmacodynamic assay to select compounds and doses for preclinical and clinical proof-of-principle studies.     

Seasonal variation in pituitary gonadotropin in the adult male newt, Cynops pyrrhogaster pyrrhogaster, revealed by isoelectric focusing technique and radioreceptor assay

The seasonal variation in pituitary gonadotropin in the adult male newt, Cynops pyrrhogaster pyrrhogaster was investigated by means of isoelectric focusing (IEF) coupled with radioreceptor assay (RRA), which employed Anolis or Xenopus testicular homogenates as receptors and 125I-rat FSH as radioligand. In the Anolis RRA system, the standard curve was obtained with 0.125-16 ng/tube of NIAMDD rat FSH I-3. Purified preparations, chicken LH IEF-1, chicken FSH AGCHD11113A and bullfrog basic gonadotropin-IV competitively inhibited the binding of the radioligand, but NIAMDD rat LH I-4 and human chorionic gonadotropin did not crossreact. The autoradiographic study revealed that 125I-rat FSH bound to the constituent cells of the seminiferous tubules in the Anolis testis, but scarcely to Leydig cells. In the IEF pattern of gonadotropin in February obtained by Anolis RRA, distinct peaks were observed at pH 9.05 (component B) and 8.55 (component C), and less distinct peaks were observed at pH 9.80 (component A), 7.55 (component D) and 7.05 (component E). When the same fractions were assayed by Xenopus RRA, five components were found in the alkaline region, which corresponded to those observed with Anolis RRA. Similar results were obtained with pituitary extracts in May. In July, the IEF pattern obtained by Anolis RRA indicated two additional components at pH 6.30 (component F) and 5.27 (component G) in the acidic region, which were not found by Xenopus RRA. The relationship between the testicular function and the nature of pituitary gonadotropin in the reproductive cycle was discussed.     

Use of a radioreceptorassay (RRA) for human luteinizing hormone/chorionic gonadotropin (hLH/CG) for detection of early pregnancy and estimation of time of ovulation in macaques

A radioreceptorassay (RRA) for macaque luteinizing hormone (LH)/chorionic gonadotropin (CG) was adapted from the clinical RRA for human LH/CG, Biocept-G?, for the purposes of detection of pregnancy prior to day 20 of gestation and for estimation of the time of ovulation in macaques. The 90-min assay procedure was simple, accurate, and reliable. Seventy-five rhesus monkeys (Macaca mulatta) and 20 crab-eating monkeys (Macaca fascicularis) were tested for the presence of CG in the serum on estimated days 17–20 of pregnancy. Of a total of 160 tests, four false negative and 0 false positive tests were obtained, for an accuracy of 97.5%. The preovulatory LH peak was detected in 19 rhesus monkeys by semiquantitative RRA of LH/CG. Ovulation was confirmed in these 19 animals by the presence of a fresh corpus luteum at laparotomy 2–10 days after ovulation, collection of an embryo, pregnancy, or subsequent cycle history. The short, simple assay procedure and the low inter-and intraassay coefficients of variation (7.3 and 3.7%, respectively) allow use of this assay in an economical, predictive, as well as retrospective, capacity for estimation of the time of ovulation in rhesus monkeys. The sensitivity, reliability, species nonspecificity, simplicity, and rapidity of performance of this RRA for LH/CG are features which add up to a useful new management tool for breeding macaques for research purposes.     

Evaluation of a radioreceptor assay to assess exogenous estrogen activity in serum of patients with breast cancer.

A radioreceptor assay (RRA) for the determination of total estrogen activity, was set up and used to assess the possible presence of exogenous molecules with estrogen activity in serum; a comparison was made with the specific radioimmunoassay (RIA) for the endogenous estrogen 17-B estradiol (17-B-E2). The assay was first performed on sera from healthy people taking estrogens in the form of oral contraceptives or lotions for local application whose total estrogenic activity in the blood was assumed to be abnormal. The assay was then performed on serum from 98 patients with early breast cancer and 20 patients with metastasis, not undergoing hormone therapy. A higher estrogen activity was found in 2.5% of sera compared to the activity found using the RIA method which is specific for endogenous estrogen 17-B-E2, the RRA/17-B-E2 ratio being higher than 3. Increased estrogen activity was found in 10% serum samples from digoxin treated cardiopathic patients, with an RRA/17-B-E2 ratio ranging from 4.4 to 20. The RRA assay could prove useful for showing up exogenous estrogen activity from various sources (drugs, food) in sera of people in whom estrogen stimulation could be potentially dangerous (i.e. in patients with hormone-sensitive tumors). This exogenous activity could support a certain degree of neoplastic stimulation and, therefore, unfavourably condition the patients' therapeutic response.     

A radioreceptor assay for insulin: direct measurement of dog pancreatic vein serum insulin.

A simple radioreceptor assay for insulin rat liver membranes as receptor sites, with sufficient specificity precision, and sensitivity to detect 10 ng or 276 muU/ml of serum insulin, has been developed. In the presence of standard porcine insulin at the concentration of 1.0 ng/tube, approximately 8% of 125I-porcine insulin was bound to the plasma membranes and ninety-five per cent of this binding was inhibited by 1.0 microgram of standard insulin per tube. Four animal insulins inhibited the binding of 125I-insulin while ACTH, glucagon, human growth hormone, and oxytocin were inert. Insulin values in dog pancreatic vein sera obtained during and after glucose loading and measured by the present radioreceptor assay agreed well with immunoreactive insulin. The ratio of IRI to the measurement by radioreceptor assay was 1.09 +/- 0.18 for the same sera.     

大鳞大马哈鱼垂体生长激素的分离、纯化与鉴定

应用ConA-Sepharose 4B亲和层析、凝胶过滤及离子交换层析等技术从大鳞大马哈鱼(Oncorhynchus tshawytscha)垂体中分离纯化了具有生物活性的生长激素(sGH)。用放射受体测定法(RRA)检测sGH组分的生物活性,结果表明纯化的8GH制品具有与兔肝细胞GH受体结合的生物活性。用放射免疫测定法(RIA)和酶联免疫吸附测定法(ELISA)分别检测了另两种垂体激素-催乳激素(PRL)和促性腺激素(GTH)在纯化的sGH制品中的残留量均在0.5％以下。用SDS-聚丙烯酰胺凝胶电泳SDS-PAGE评价sGH制品的电泳纯度并测定了其分子量为22000左右。等电聚焦电泳表明该种鱼GH由等电点分别为6.3和6.6的两种形式的分子组成。     

A novel and sensitive radioreceptor assay for serum melatonin levels.

A simple and sensitive radioreceptor assay (RRA) has been developed to measure melatonin levels in serum. The assay is based on competition between 2-[125I]iodomelatonin ([125I]MEL) and melatonin for binding to high-affinity binding sites in chick forebrain. To measure the amount of melatonin present in a serum sample, it was extracted with dichloromethane and added to the assay medium. The percentage inhibition of radioligand binding in the presence of the extracted serum was determined and compared to the percent displacement by known amounts of melatonin in a standard curve. There was little or no cross-reactivity with other structurally related compounds. The sensitivity of the assay is approximately 1.5pg/0.15 mL and the intra- and inter-assay variations are approximately 8%. Since the RRA results are comparable to that of an established radioimmunoassay (RIA), it provides a sensitive and rapid alternative to the more time consuming RIA.     

Radioreceptor assay for lactogenic hormones based on membranes from rat mammary tumour

A highly sensitive radioreceptor assay (RRA) for human prolactin (hPRL) based on membrane preparations obtained from chemically induced rat mammary tumour is described. The binding of 125I-labelled, highly purified pituitary human prolactin was specific for lactogenic hormones and depending on time, temperature, and concentration of receptor protein. Optimal specific receptor binding (18-20%) was obtained by incubation at 21 degrees C for 18 h. The prolactin receptor was shown to have a single "class" of binding sites with an affinity constant (Ka) of 6.0 X 10(10) mol-1. The binding capacity was 8-33 fmol/mg membrane protein. The sensitivity of the radioreceptor assay was 0.5 ng/ml ovine prolactin (NIH-PS-10) or 0.84 ng/ml human prolactin (NIH-VLS-4). The receptor binding activity of various purified prolactin preparations from different species was comparable to the biological hormone activities, indicating that this in vitro assay system measures values which are biologically relevant.     

Zebra chorionic gonadotropin: partial purification and characterization

Six samples of pregnant zebra (z) serum from the first and second trimesters of pregnancy were analyzed by RIA and shown to have chorionic gonadotropin levels comparable to that of the mare (0.9-5.3 micrograms/ml); first trimester levels in most cases were higher than second trimester levels. A pool of the sera (10 ml) was fractionated by methods previously employed for the purification of equine (e) and donkey (d) chorionic gonadotropin to achieve a concentration of the zebra chorionic gonadotropin (zCG). A yield of 1.0 mg of glycoprotein was obtained. HPLC analysis of the material indicated the content of zCG to be about 7%. Its molecular size as judged by Ve/Vo values is smaller than eCG, greater than ovine LH, and about the same as equine LH. The zCG was tested in RIAs for LH and eCG, radioreceptor assays (RRA) for LH and FSH, and the rat testis Leydig cell assay for LH. Comparisons were made with equine and donkey chorionic gonadotropin, and equine and zebra LH. The results, preliminary because the preparation is not of high purity, showed that zCG is bioactive as an LH; immunologically similar to eCG, eLH, dCG, and zLH; and competes in RRAs for LH but not FSH receptors. It differs, therefore, from eCG and eLH--which have high levels of intrinsic FSH activity, and is more like dCG, dLH, and zLH--all of which have minimal if any FSH activity.     

Characterization and detection of naturally occurring antibodies against IL-1 alpha and IL-1 beta in normal human plasma.

During the development and testing of a radioreceptor assay (RRA) for human IL-1, we have detected and identified the presence of auto-antibodies to IL-1 in normal human plasma (NHP). The RRA is based on the competition between human 125I-labeled rIL-1 alpha and standard or unknown quantities of IL-1 alpha or IL-1 beta for binding to a limited amounts of IL-1 receptor (IL-1R) isolated from the EL4 mouse thymoma cell line. NHP from 20 out of 100 unselected blood donors were found to completely inhibit the binding of 125I-labeled IL-1 alpha to its receptor, suggesting the presence in these NHP samples of either abnormal amounts of IL-1 or of a factor binding to the 125I-labeled IL-1 alpha. Special care was taken to ascertain that the inhibitory factors were antibodies and not soluble IL-1 receptor antagonist. When plasma samples with inhibiting activity were incubated with labeled IL-1 alpha and chromatographed on a Sephadex G200 column, they were found to contain 125I-labeled complexes with an apparent molecular weight of 150-200kD. The IL-1 binding factor could be eliminated from plasma by incubation with protein A-Sepharose, suggesting that it consisted in IgG antibodies directed against IL-1. Furthermore, the antibody nature of the inhibiting factor was confirmed by its binding to purified rIL-1 coupled to Sepharose. Screening of 200 NHP samples by incubation with 100 pg of 125I-labeled IL-1 followed by precipitation with 12% of polyethylene glycol (PEG) confirmed that about 25% of NHP contain detectable IgG antibodies to IL-1 alpha, while only 2% of NHP contain antibodies to IL-1 beta. No correlation between the presence of these anti-IL-1 antibodies and any particular major histocompatibility complex or any pathological conditions was detected. We suggest that all serum samples assayed for IL-1 alpha or IL-1 beta content should be pretested with the PEG precipitation assay described here.     

Effects of single and multiple injections of ketamine hydrochloride on serum hormone concentrations in male cynomolgus monkeys

Using simulated short- and long-term effect studies, we evaluated the effect of ketamine anesthesia on serum cortisol, testosterone, and immunoreactive luteinizing hormone (ILH) and bioactive LH (BioLH) concentrations in adult male cynomolgus monkeys. Cortisol, testosterone, and ILH were measured by use of radioimmunoassay, and BioLH was measured by use of a radioreceptor assay method. For the acute effect, the first group (eight monkeys) was given four successive intramuscular injections of ketamine (10, 5, 5, and 5 mg/kg of body weight at 0, 30, 60, and 110 min respectively). Blood samples were taken at 0, 15, 30, 45, 60, and 120 min. For the long-term effect, the second group (10 monkeys) was given a single injection of ketamine (10 mg/kg) once a week for 4 consecutive weeks. Blood samples were taken 5 to 10 min after each injection, then were used to determine the variation in hormone concentrations among the monkeys (inter-individual variation) and within each monkey (intra-individual variation). There were no statistically significant differences in serum cortisol, testosterone, ILH, and BioLH values between the first blood sample (before the ketamine injection) and sequential blood samples in monkeys of the first group. Although intra-individual variation in the hormones (i.e., hormonal change within each monkey) was not statistically significant, inter-individual variation (among the monkeys) was significantly (0.00001 < P < 0.033) different in monkeys of the second group. These results indicate that an adequate number of animals must be used to minimize animal-to-animal variability. Our results confirm that ketamine is a suitable anesthetic agent to immobilize male cynomolgus monkeys in experimental studies (short- and long-term studies) aimed at elucidating hormonal changes.     

Demonstration of potent, gonadotropin-like biological activity in the serum of rats during midpregnancy

The gonadotropin-like activity (GnLa) of serum from pregnant rats was measured using the rat interstitial cell testosterone (RICT) bioassay. Serum GnLA was elevated on day 9 of pregnancy, peaked at 7.2 μg rat LH-RPl equivalents/ml on day 11 and declined to undetectable levels by day 15. Serum LH, measured by homologous RIA, was consistently low (<20 ng/ml) during pregnancy, except near term.Rat placental lactogen (rPL), which was measured in the same serum samples by rat radioreceptor assay (RRA), reached maximal concentrations on days 12 and 13 of pregnancy.These data suggest the presence in pregnancy serum of a potent-gonadotropin-like hormone, different from pituitary LH, whose origin is unknown. Furthermore, there are discrepancies between the times of appearance of this GnLA and rPL.     

125I-LH binding to rat testes at various ages and posthypophysectomy.

Binding of 125I-LH by the rat testes was investigated during various stages of sexual maturation and in mature animals following hypophysectomy. Hormone binding per mg testicular tissue increased with age and was shown to be due to larger receptor concentration rather than greater binding affinity. This observation cannot be accounted for by changes in the relative number of Leydig cells and suggests, therefore, that Leydig cells acquire additional receptors during sexual maturation. Binding of 125I-LH to mature testes declined after hypophysectomy. Three days following pituitary removal the LH-receptor concentration decreased to one half of normal control value, then remained unchanged until the 37th post-operation day. Replacement therapy with LH, FSH or testosterone propionate failed to maintain 125I-LH binding at prehypophysectomy level.     

Measurement of human FSH by radioreceptor assay

We established a sensitive RRA system for human FSH, employing PMS-treated immature rat ovary. The dissociation constant of the binding of the receptor preparation to NIAMDD human FSH-2 was 1.15 x 10(-10) M. The standard curve was obtained with 0.2-25.6 ng/tube of NIAMDD hFSH-2. Purified hLH, hTSH, and HCG had no significant effect on the binding. When the anterior pituitary homogenates obtained from humans were assayed by this system, the intra-assay and inter-assay coefficients of variation were 11.9% and 13.4% respectively, and the assay values correlated well with those obtained by RIA. This assay is applicable for the measurement of FSH in serum, when the non-specific inhibitor effects of serum are compensated for by the addition of merthiolate and when FSH-free serum is used instead of the buffer for the standard curve. The intra-assay and inter-assay coefficients of variation were 9.31% and 19.7% respectively. The assay values correlated with those obtained by RIA under the same physiological state. The ratio of the assay values RRA/RIA, was dependent upon the physiological state, e.g. 6.29 in men, 3.84 and 4.18 in women at follicular and luteal phase respectively and 2.40 in menopausal women. During the menstrual cycle, our results showed that the value of RRA/RIA derived from serum did not change significantly.     

Discrimination of a colony stimulating factor subclass by a specific receptor on a macrophage cell line

Utilizing the high affinity interactions between pure ¹²⁵I-L cell colony stimulating factor and its receptor(s) on the murine macrophage cell line J774, a murine radioreceptor assay (RRA) has been developed. The murine RRA selectively detects a colony stimulating factor (CSF) subclass (CSF-1) previously defined by murine radioimmunoassay (RIA) (E.R. Stanley, Proc. Nat. Acad. Sci., USA, 76:2969–2973 ('79)). CSF-1 stimulates macrophage production exclusively, and the occurrence of the CSF-1 receptor(s) appears to be restricted to cells of the mononuclear phagocytic system (L.J. Guilbert and E.R. Stanley, J. Cell Biol. 85:153–160 ('80)). The murine CSF-1 RRA failed to detect a variety of other CSF subclasses, growth factors, and hormones. In contrast to data obtained with the murine CSF-1 RIA, human CSF-1 (e.g., human urinary CSF) is detected by the mouse CSF-1 RRA almost as sensitively as murine CSF-1. In addition, there was an absolute correlation between CSF-1 levels determined by murine CSF-1 RRA and those determined by a human CSF-1 RIA for a variety of human CSF-1 sources. The murine CSF-1 RRA is a sensitive (sensitivity 5 units or 1.0 femtomole of CSF-1 protein), rapid, and highly specific assay for CSF-1 in both murine and human sources.     

Dynamic change in charge heterogeneity of pituitary FSH throughout the estrous cycle in female rats

Anterior pituitaries were removed from female rats at various stages during the estrous cycle and FSH was fractionated by isoelectric focusing (IEF). Fourteen FSH components were observed during the estrous cycle and twelve of them were distributed between pH 3.71 and 6.66. IEF profiles of FSH in the pituitaries varied with the stage in the estrous cycle. Especially at the time of serum FSH surge on the day of proestrus, most of the components decreased, while only a highly alkaline component showed an increase. When these FSH components were separated and their nature was examined by radioreceptor assay (RRA), radioimmunoassay (RIA) and gel filtration, differences were observed among these components in the RRA/RIA ratio and gel filtration profile. As a general tendency, the RRA/RIA ratio of the components became greater while the apparent molecular size became smaller, as their pI became higher. However, some highly acidic components showed a biphasic elution pattern and the most acidic one eluted the latest on gel filtration, suggesting that these components may be heterogeneous in terms of molecular size. The FSH concentration in sera collected at different stages in the estrous cycle was measured by both RRA and RIA. The RRA/RIA ratio was high when the serum immunoreactive FSH was low, and low during the FSH surge. From these findings, it is concluded that the quality of FSH molecules present in the anterior pituitary gland changes dynamically throughout the estrous cycle, especially during the period of serum FSH surge. Furthermore it is suggested that the type of FSH secreted from it also varies according to the stage in the estrous cycle.