Folia Endocrinologica Japonica
Online ISSN : 2186-506X
Print ISSN : 0029-0661
ISSN-L : 0029-0661
Volume 54, Issue 10
Displaying 1-8 of 8 articles from this issue
  • Takara YAMAMOTO, Kazuo OTSUBO, Kazuya OSHIMA, Takao YAMAMOTO, Hideo HO ...
    1978 Volume 54 Issue 10 Pages 1103-1115
    Published: October 20, 1978
    Released on J-STAGE: September 24, 2012
    JOURNAL FREE ACCESS
    Estradiol-17β (E2) has recently been reported to be conjugated into its glucosiduronate or sulfate in human or baboon kidneys in vivo or in vitro. To investigate whether glucosiduronation or sulfation of E2 occurs in the dog kidney in vivo and to clarify the fate of conjugated E2 in the kidney, this experiment was performed using the following techniques.
    1) 3H labeled unconjugated or conjugated E2 was, injected into one of the renal arteries and 14C labeled unconjugated E2 was simultaneously injected into a peripheral vein of a dog (shown as Fig. 1).
    2) Following the injection, urine from both sides was collected separately through ureterostomies at various time intervals over a period of 7 hours.
    3) Aliquots of the collected urine samples were counted for radioactivity.
    4) The metabolites in the collected urine samples were identified on the DEAE Sephadex A-25 column chromatography, enzyme hydrolysis, and thin layer chromatography, using two different solvent systems : chloroform-ethyl ether, 1 : 4 and cyclohexane-ethyl acetate, 1 : 1.
    In the first experiment, the simultaneous injection of (6, 7-3H) E2 into a right and (4-14C) E2 into a peripheral vein, the excretion of 3 H labeled compounds in the injected side was about five times greater than it was in the opposite side in the early interval (for 15 min. following the injection), and the 3H/14C ratio in the injected side was about ten times what it was in the initial ratio. Urine samples in the injected side in the early interval were mainly composed of 3H labeled unconjugated E2 (shown as Fig. 3). In all experimental time intervals, 14 C compounds were excreted at almost equal percentages in both sides for 7 h. following the injection (shown as Fig. 2). When (6, 7-3H) E2-3G or (6, 7-3H) E2-17G was injected into a renal artery, it was also excreted directly through the kidney without any change in the form in the early intervals (shown as Fig. 5 and Fig. 6). Metabolites in the urine samples in the late intervals were identified as estrogen glucosiduronates (E1-3G and E2-3G) and estrogen sulfates (E1-S and E2-17α-S) following the above procedures, and E2 was changed to E1 in small amounts. Accordingly, glucosiduronation did not occur in dog kidneys in vivo, and these metabolites were thought to have occurred in the systemic circulation.
    In the second experiment, the simultaneous injection of (2, 4, 6, 7-3H) E2 or (2, 4, 6, 7, 16, 17-3H) E2 into a renal artery and (4-14C) E2 into a peripheral vein, the total excretion of 3H compounds (34% or 46% on an average following a renal injection of (2, 4, 6, 7-3H) E2 or (2, 4, 6, 7, 16, 17-3H) E2, respectively) was higher in comparison with the first experiment (14% on an average following a renal injection of (6, 7-3H) E2). The 3H/14C ratios in the second experiment were about ten times that of the first experiment (shown as Table 2). Therefore, E2 was thought to be metabolized at the 6, 7 position in the systemic level.
    As shown above, the metabolism of unconjugated or conjugated E2 in the dog kidney in vivo and metabolism at the 6, 7 position of E2 in the systemic level were clarified in these experiments.
    Download PDF (1110K)
  • Part I : Plasma Secretin Concentration and Pancreatic Exocrine Secretion after Intravenous Secretin or Intraduodenal HCl and 1-Phenyl-1-Hydroxy-n-Pentane Administration
    Makoto OTSUKI, Choitsu SAKAMOTO, Mitsuo MAEDA, Hosai YUU, Tomio YAMASA ...
    1978 Volume 54 Issue 10 Pages 1116-1124
    Published: October 20, 1978
    Released on J-STAGE: September 24, 2012
    JOURNAL FREE ACCESS
    A demonstration of the greater effect of oral glucose administration, as compared with intravenous administration, in stimulating a rise in serum insulin levels led to the hypothesis that during the passage through the gastrointestinal tract ingested glucose induces the release of intestinal factors which augment the beta-cytotropic effect of glucose. Recent work has suggested that secretin, a hormone which can be extracted from duodenal mucosa, has a significant insulinotropic effect in vivo and in vitro.
    Intestinal acidification is presently the only recognized stimulus for the secretion of secretin. However, widely different results have been obtained by several laboratories concerning the beta-cytotropic action of endogenous secretin. Furthermore, overestimation of secretin release calculated on the basis of pancreatic bicarbonate secretion after intraduodenal instillation of HCl may indicate that intestinal acidification causes a release of not only secretin but also other stimulators of pancreatic exocrine and endocrine secretion.
    In the present study we investigated the effect of intraduodenal infusion of HC1 or 1-phenyl-1-hydroxy-n-pentane (PHP) on the concentration of immunoreactive secretin in portal plasma and pancreatic exocrine secretion, and compared it with that of intravenous synthetic secretin to further evaluate the beta-cytotropic effect of endogenous secretin. In addition, the effect of somatostatin on HCl-stimulated secretin and pancreatic exocrine secretion was investigated.
    Male Wistar rats weighing 250-300g and fasted overnight were used in all experiments. Under pentobarbital anesthesia, the abdomen was opened through a midline incision. A polyethylene catheter was inserted into the bile duct at the proximal end to drain out the bile. For measurement of pancreatic exocrine secretion, a calibrated capillary tube was attached to the free end of the pancreatic cannula which was inserted into the distal end of the common bile duct at a point shortly before it entered the duodenum. Every 10 min, the capillary tube was replaced and the rate of flow of the pancreatic juice down the tube was measured for an hour. A sample of the juice was diluted with physiological saline of 500μl and stored at-20°C until amylase assay. Blood samples were obtained through a polyethylene catheter placed in the portal vein.
    Infusion of the duodenum was accomplished by a catheter inserted through an incision in the stomach into the duodenal bulb and kept in place by means of a tight ligature around the pylorus. Care was taken not to damage or ligate the gastroepiploic vessels. The following solutions were constantly introduced into the duodenum for 2 min (2ml/min) : 0.1N-HCl, 200mg/kg/2ml PHP, and 2.5% arabic gum solution (placebo).
    Synthetic secretin, dissolved in isotonic saline, was infused into the jugular vein for 20 min with a syringe pump at a rate of 10ng/kg/min. Somatostatin was dissolved in saline and administered intravenously by a syringe pump for 30 min, starting 10 min before the intraduodenal instillation of HC1 and continuing for a further period of 20 min (50μg/kg/h). Plasma secretin concentrations were determined by radioimmunoassay using the double antibody method. Amylase activity in the pancreatic juice was assayed by the chromogenic method using blue-dyed starch polymer.
    Both 0.1N-HC1 and the 200mg PHP infusion elicited a rapid immunoreactive secretin (IRS) increase, reaching a peak value (HCl : 374.6 ± 26.3, PHP : 335.4 ± 68.9pg/ml) at 5 min. An acid load of 0.4mEq resulted in pancreatic amylase release about 12 times higher than the value obtained with the PHP instillation and synthetic secretin infusion (HCl : 2130.2 ± 250.9, PHP : 173.5 ± 22.8, intravenous secretin : 281.9 ± 62.3 Somogyi Units/10 min), though the portal venous IRS levels and the flow rate of the pancreatic juice were nearly the same.
    Download PDF (877K)
  • Tomohiko HONDA
    1978 Volume 54 Issue 10 Pages 1125-1150
    Published: October 20, 1978
    Released on J-STAGE: September 24, 2012
    JOURNAL FREE ACCESS
    It is well recognized that the purely clinical approach does not always detect every compromised fetus. Laboratory tests for placental function is now regarded as part and parcel of the antenatal care of the patient with high-risk pregnancy. The reliability of these tests as a screening procedure for risk pregnancy is fairly well established at present.
    For example, the determination of estrogen (estriol) in maternal urine and hCS in maternal blood is clinically most useful. “Useful” in this sense, however, does not imply that they are intrinsically superior to other parameters, but merely implies that the technology is well established and that there is adequate published information regarding it. Therefore, the author has attempted to set up a practical guideline in screening the function of the feto-placental unit by measuring hCS with hemagglutination assay (HPL-HAR Test kit) and determining estriol by hemagglutination inhibition assay (E3-HAIR Kit).
    Urinary estriol levels were plotted on the vertical axis, and serum hCS was plotted on the horizontal axis. Low estriol and hCS zones were determined by drawing a line at the lowest limit of these hormones in normal pregnancy. The area on which the two zones were superimposed was designated an absolutely abnormal zone. Furthermore, dehydroepiandrosterone sulfate (DHAS) and oxytocin challenge tests were added for a dynamic test of placental function.
    In early pregnancy, placental function was estimated by the measurements of hCG and hCS, the hormones produced by the placenta. hCG levels were plotted on the vertical axis, and hCS values were plotted on the horizontal axis of the monitoring table.
    Summarized data are as follows :
    In threatened abortion which terminated in fetal loss, “the point” moved to the lower left part on the “table”. On the other hand, in cases with favorable outcome, “the point” moved to the right. In molar pregnancy, the hormone pattern was 'high hCG' and 'low hCS' and was different from that in abortion. In cases of anencephalic, congenital enzymatic deficiency of the placenta, and Wharton's jelly defect of the umbilical cord, “the point” remained in the low estriol zone. With mild toxemia of pregnancy, “the point” also remained in the low estriol zone. With sensitized rhesus incompatibility, “the point” gradually moved to the low estriol zone. In cases of placental insufficiency and mild toxemia of pregnancy (hypertensive type), “the point” moved to the low hCS zone. In cases of chronic hypertension, retarded fetal growth, and aortic insufficiency, “the point” remained in the low hCS zone. With intrauterine fetal death, “the point” was always in the absolutely abnormal zone. With severe toxemia of pregnancy, “the point” moved into the absolutely abnormal zone. In cases of well-controlled diabetic disease or hyperthyroidism, and of rhesus incompatibility with low antibody titers, “the point” was in the normal zone.
    In cases with “the point” present in the low estriol zone, the DHAS dynamic test should be done, and the reserve-function of the feto-placental unit should be evaluated. In cases with “the point” present in the low hCS zone, it is necessary to use the oxytocin challenge test to assess the reserve-function of the placenta.
    In conclusion, the effective use of these methods is of value in predicting the dysfunction of the feto-placental unit, and it is hoped that the employment of these methods for the high risk foetus in the early stages might lead to more intensive obstetric care and might contribute to the reduction of perinatal loss.
    Download PDF (1732K)
  • Part I. A comparative study of impurities in commercially available thyroid preparations
    Nagao HEKI
    1978 Volume 54 Issue 10 Pages 1151-1156
    Published: October 20, 1978
    Released on J-STAGE: September 24, 2012
    JOURNAL FREE ACCESS
    The conversion process of T4 to T3 or reverse T3 presumably involves deiodinase enzyme systems capable of removing iodide molecules at the 5 or 5′ positions. Although there are scant data to indicate whether these metabolites (T3 and r-T3) themselves are capable of undergoing further deiodination reactions, it seems likely on theoretical grounds that their continued exposure to operative deiodinase enzyme systems would result in the formation of other less iodinated metabolites, such as 33′-T2.
    The present report describes the development of a simultaneous analysis of thyroid analogues in biological fluids by mass fragmentography using GC-MS. Analysis was carried out on serum and urine from normal subjects. Similarly, analysis was performed on commercially available thyroid preparations. The TMSi derivatives of the compounds were analyzed by the GC-MS system equipped with a 3ft X 3mm column packed with 1% OV-1, and the temperature was programmed from 250 to 330 C at 10 C/min increments. Thyroid fractions thus separated on the column were subjected to detection of 33′-T2 or r-T3. The following results were obtained.
    1) DL-35T2 (commercially available preparation) was found to be composed of 7 mass-spectrometrically different fractions which were detected at the Tyrosine zone, MIT zone, DIT zone, decomposed product of 35-T2 zone, 35-T2, post 35-T2 zone (33′-T2) and leading edge of 33′-T2 zone (decomposed product of 33′5-T3), respectively.
    2) L-33′5T3 (commercially available preparation) was found to be composed of 6 mass-spectrometrically different fractions which were detected at the Tyrosine zone, MIT zone, DIT zone, 35-T2 zone, 33′5-T3 zone and post 33′5-T3 zone (33′5′-T3 = r-T3), respectively.
    3) The ability to simultaneously analyze 33′-T2, r-T3 and other thyroid analogues should be effectively utilized in order to study further their physiology, production and clearance.
    Download PDF (530K)
  • Part II. Mass fragmentographic identification of 33′-T2 and r-T3 in biological fluids
    Nagao HEKI
    1978 Volume 54 Issue 10 Pages 1157-1162
    Published: October 20, 1978
    Released on J-STAGE: September 24, 2012
    JOURNAL FREE ACCESS
    In view of the previous report of the possibility of 33′-T2 and r-T3 measurements, the identification of these iodinated compounds was performed by mass fragmentography. The peaks at m/e 218,624 (33′-T2) and m/e 218,750 (r-T3) were applied to establish the precise qualitative evaluation of unknown peaks. The base peak at m/e 218 (thyroid analogues) was applied to avail itself of simultaneous multiple ion analyses by GC-MS. The following results were obtained.
    1) Mass fragmentography makes possible the simultaneous measurements of 33′-T2, r-T3 and other thyroid analogues with 1 ml of serum and urine samples obtained from rats or human beings.
    2) This method is very convenient for extracting the compounds from biological fluids, and the procedure can be carried out easily in a short time. The specificity of this method surpasses and cannot be compared to any other existing quantitative methods.
    Download PDF (755K)
  • Takashi HIGUCHI
    1978 Volume 54 Issue 10 Pages 1163-1186
    Published: October 20, 1978
    Released on J-STAGE: September 24, 2012
    JOURNAL FREE ACCESS
    To get more insight into the hypothalamic control of gonadotropin secretion, we attempted to define the secretory patterns of the luteinizing hormone-releasing hormone (LH-RH), by measuring hypothalamic LH-RH contents as well as by observing the effects of immunoneutralization of endogenous LH-RH on serum gonadotropin levels.
    Female rats of Wister and Fischer strain with regular 4 or 5 day estrous cycles were used. They were raised in a light controlled (from 5 : 00 to 19 : 00), air conditioned room. Concentrations of serum and pituitary gonadotropins were measured by double antibody radioimmunoassay with NIAMDD kits and anti-LH serum from Dr. G.D. Niswender. Hypothalamic LH-RH was assayed by radioimmunoassay using anti-LH-RH serum from Drs. G. D. Niswender and T. Nett and synthetic LH-RH for iodinated and standard hormones. LH, FSH and LH-RH values were expressed in terms of NIH-LH-Sl, NIH-FSH-Sl and synthetic LH-RH respectively. Blood samples were taken either by decapitation or through the indwelling intra-atrial cannula. The hypothalamus was excised with scissors and stored in 0.1N HCl at-20°C. Before assay for LH-RH, acidic extract of the hypothalmus was neutralized with 0.1N NaOH.
    In rats with 4 day cycles, serum LH levels increased with a peak at 16 : 00 to 18 : 00 on the day of proestrus, and serum FSH concentrations also increased with a longer duration than that during the LH surge. Hypothalamic LH-RH contents decreased in the late afternoon of the proestrus. In rats with 5 day cycles, serum gonadotropin and hypothalamic LH-RH changes similar to those in rats with 4 day cycles were observed on the day of proestrus. In long-term ovariectomized animals, an injection of estradiol benzoate (EB 20μg) decreased serum LH and FSH concentrations, and an additional injection of EB (20μg) or progesterone (P 2mg) 72 hrs after the first EB injection evoked an LH surge as observed in rats with normal cycles. However, hypothalamic LH-RH contents failed to change significantly during the steroid induced LH surges. Furthermore, no appreciable changes in the hypothalamic LH-RH contents were induced in the case of LH release evoked by electrochemical stimulation of the medial preoptic area (MPO) in the proestrous rats in which spontaneous LH surge had been blocked with pentobarbital (30mg/kg B.W.) injected at 13 : 00.
    Anti-LH-RH serum obtained from male rabbits immunized with LH-RH-BSA conjugate bound I125-LH-RH. Synthetic LH-RH and rat hypothalamic extract produced parallel inhibition of the binding. This anti-LH-RH serum showed cross reactivity to neither hypothalamic nor pituitary hormones examined other than LH-RH. From the cross reactivity to the LH-RH fragments, the antibody seemed to have stronger affinity to the C-terminus than to the N-terminus of the LH-RH molecule. This antiserum inhibited spontaneous gonadotropin surges and ovulation when injected at 12 : 00 on the day of proestrus in the volume of 0.5 to 1.0 ml. The cyclicity of vaginal smears was temporarily interrupted with successive leucocytic smears for 1 to 3 cycles after the antiserum injection and thereafter returned to the regular cycle. This anti-LH-RH serum inhibited the rise of serum LH and FSH concentrations induced by EB or EB and P injections. Moreover, it prevented the elevation of serum gonadotropins induced by electrochemical stimulation of the MPO or arcuate nucleus in pentobarbital-blocked proestrous rats.
    In order to elucidate the roles of LH-RH in controlling the basal gonadotropin secretion, anti-LH-RH serum was injected into long-term ovariectomized rats. Both serum LH and FSH levels lowered after the antiserum injection with short latency, but FSH levels decreased less than LH levels. In short-term ovariectomized rats, the prompt increase of FSH levels during the 6 hrs after ovariectomy was never inhibited by the anti-LH-RH injection, whereas LH concentrations were below the lowest detectable limits of the assay.
    Download PDF (2748K)
  • Hiroyuki HOSOJIMA, Nagao HEKI
    1978 Volume 54 Issue 10 Pages 1187-1197
    Published: October 20, 1978
    Released on J-STAGE: September 24, 2012
    JOURNAL FREE ACCESS
    In order to obtain more precise information on thyroid functions in renal failure, attempts were made to analyze the Hypothalamo-hypophyseal axis in the regulation of thyroid function. TRH levels and other thyroidal function tests were carried out on sera and urine from patients with renal failure and thyroid diseases. Analysis of TRH in serum and urine was performed by mass fragmentography using a GC-MS combined method.
    Fifteen out of 28 patients had enlarged thyroid glands, goiter in high prevalence. Thyroid function studies showed low serum thyroxine and triiodothyronine, and high serum thyrotropic hormone levels. Serum and urinary TRH in chronic renal failure were strikingly higher than the other thyroid diseases.
    There were negative correlations between RIA-T3, Resomat-T4 and serum and urinary TRH levels respectively. There was a negative correlation between serum TSH and serum TRH, but no significant correlation was noticed between serum TSH and urinary TRH levels. There was a high positive correlation between serum TRH and urinary TRH levels.
    From these findings, it is thought that serum and urinary TRH level values are a more reliable index for the clinical evaluation of thyroid functions in renal failure than the other sample under laboratory data. In patients with chronic hemodialysis, the prevalence of goiter is relatively high, and their thyroid functions are abnormal. However, the cause of the abnormalities is not clear. It is postulated that the possibility of a breakdown of compensation in the Hypothalamo-hypophyseal axis for the regulation of the thyroid function may be seen in chronic renal failure.
    Download PDF (862K)
  • Teruhiko TAMAYA, Norio FURUTA, Toshihiko MOTOYAMA, Yousuke OHONO, Shin ...
    1978 Volume 54 Issue 10 Pages 1198-1206
    Published: October 20, 1978
    Released on J-STAGE: September 24, 2012
    JOURNAL FREE ACCESS
    The present study was designed to determine the characteristics of the progesterone receptor and chromatin binding site (“acceptor”) of the progesterone-receptor complex in the rabbit uterus. The uterus was obtained from an estrogen-primed immature female rabbit. The binding of progesterone to the uterine receptor was examined in vitro. The progesterone-receptor binding was reduced only by proteases, and phosphorus moiety may not be related for progesterone-receptor binding.
    The effects of enzymes on the acceptor of the chromatin were investigated. The progesterone-receptor complex was bound to the dehistonized chromatin. The dehistonized chromatins, which were pretreated with enzymes at 4°C or 37°C for 30 minutes, were incubated with 3H-progesterone prelabeled uterine cytosol at 4°C for 30 minutes, and the radioactivity in the chromatin pellet was counted.
    Proteases effectively decreased the receptor binding capacity to the dehistonized chromatin in the following order : pronase>trypsin>papain>α-chymotrysin. DNAse moderately and phospholipase A slightly decreased its binding capacity. The results may indicate that the acceptor site of the progesterone receptor is nonhistone protein over DNA of chromatin and may contain phosphorus moiety.
    Download PDF (743K)
feedback
Top