The rapid calcium infusin test (Goldsmith, R.S. et al. 1962) was evaluated in 3 normal subjects, 6 patients with parathyroid disorders, 12 patients with urolithiasis and 12 patients with various other diseases. Twenty-five milliliter of 2% calcium chloride solution was infused intravenously over a ten-minute period starting at 9 a.m. and the response of urinary phosphate excretion to calcium infusion was investigated for 3 hours.
All normal subjects, 10 patients with urolithiasis and 8 patients with various other diseases responded to calcium infusion, but a patient with hyperthyroidism and 2 patients with urolithiasis showed delayed responses to calcium infusion. The phosphaturic rhythm was unaffected by calcium infusion in a patient with renal tubular acidosis, a patient with Fanconi's syndrome, 3 hypoparathyroid patients and a patient with multiple myeloma who had severe impairment of renal function. The abnormal spontaneous phosphaturic rhythm, i.e. a progressive fall in phosphorus excretion, was observed in a patient with acromegaly and a patient with hypercalcemia of unknown etiology under corticosteroid therapy. A hyperparathyroid patient associated with adult Fanconi's syndrome showed a decreasing Up/Ucr ratio following calcium infusion, though his spontaneous pattern was not ascertained.
The fall in urinary phosphorus excretion below the control level within 3 hours after calcium infusion may be considered as a normal response to the rapid calcium infusion test, when calcium chloride is infused instead of calcium glucoheptonate.

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Testosterone propionate (TP) was injected subcutaneously to pregnant rats at the later stage of gestation, and its effect on the differentiation of sexual functions of the offsprings were studied. The day in which sperm or plug was found in vaginal smear was designated as the 1st day of pregnancy. The successive injections were carried out once daily for 7 days from the 15th to the 21st day of pregnancy. The single injections were carried out on the 15th, 18th or 21st day of pregnancy. The dosages of TP were 0.05, 0.1, 0.5 and 5.0mg per pregnant rat a day in the case of successive injections and 5.0 and 10.0 mg per rat in the single injection. The results are summarized as follows :
(1) Except in only one rat receiving 5.0mg a day of TP, no parturition delay was observed in the pregnant rats injected with TP successively. The rats receiving the single injection of TP, however, demonstrated parturition delay when the administration was done on the 15th or the 18th day of pregnancy.
(2) Fetal resorbing activity of TP was observed in accordance with the increase of dosages in the successive injections and in the single injection on the 15th or the 18th day of pregnancy.
(3) The masculinizing effect of prenatal TP, administered either by single injection on the 18th day or by successive injections from the 15th to the 21st day of pregnancy, on the female fetuses was demonstrated in the ano-genital distance (AGD) of newborns on the day of their birth. The intensity of this effect was elevated with the increase of the dosages in successive injections. No remarkable change was observed in the AGD of male newborns.
(4) The prenatal effect of TP on the female AGD were still retained and rather extended even on the day of weaning, when administered by the successive injections at the dosages of 0.5mg a day or more or by the single injection on the 18th day of pregnancy. A single injection of 5.0 or 10.0mg TP on the 21st day of pregnancy did not affect the AGD of female newborns on the day of their birth, but gradually produced a masculinizing effect on female young and gave a significant enlargement of AGD when compared with the control on the day of weaning.
(5) The prenatal treatment of TP, 0.5mg a day for 7days (Day 15 to Day 21) or 5.0 or 10.0mg on the 21st day of pregnancy, produced not only delayed development of external genitalia of the young, but also caused abnormality in the female genitals. Female young had no vaginal openings, when their mother had received 5.0mg a day of TP from the 15th to the 21st day or 5.0 or 10.0mg of TP on the 18th day of pregnancy.
These results suggest that TP administered in latepregnancy not only influences directly the fetal genitals to cause masculinization of female offspring, but also affects the undifferentiated central nervous system of fetuses to produced delayed development or interruption of sexual maturity and function.

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Prenatal effects of testosterone propionate (TP) upon the postnatal sexual functions of rat youngs were studied. The steroid dissolved in sesame oil was administered subcutaneously into pregnant Wistar rats once daily for 7 days from the 15th to the 21st day of pregnancy or with a single injection on the 15th, 18th or 21st day of pregnancy. The dosages of TP were 0.05, 0.1, 0.5 and 5.0 mg per pregnant rat a day in the case of successive injection and 5.0 and 10.0 mg per rat in the single injection. The results obtained are summarized as follows :
(1) The female offsprings of the rats treated with TP either in the successive injection of 0.05 or 0.1 mg a day, or in the single injection on the 15th day of pregnancy showed the same normal 4-days estrus cycles as the controls. However, a higher dosage in the successive injection, 0.5 mg a day of TP, or a later administration on the 21st day of pregnancy, produced persistent vaginal cornification in the female offsprings, which had abnormal, masculinized external genitaliae with the vaginae never opened beyond a narrow external orifice. Female offsprings had no vaginal openings, when their mother had received 5.0 mg a day of TP from the 15th to the 21st day or 5.0 or 10.0 mg of TP at the 18th day of pregnancy. Moreover, persistent cornification of the vagina induced by the prenatal TP was not interrupted with a single subcutaneous injection of progesterone.
(2) The mating tests with an intact male partner indicated that the copulatory ability of the female offsprings showing normal estrus cycles seemed to be normal, but that of the females showing persistent vaginal cornification was heavily damaged after the prenatal medication of TP. Although the male offsprings of the rats receiving prenatal TP had macroscopically normal external genitalia, their copulatory ability tested by mating with an intact female partner was also clearly reduced especially in the case of successive injection of TP into the pregnant mothers. Theseresults suggest that the reductions of the copulation rate in both sexes born from the mothers treated with TP may be due to the central effect of the steroid on the fetal brain rather than the peripheral effect on the external genitalia of fetuses.
(3) The rats receiving the prenatal TP treatment were autopsied at 130 days of age. The data on the females indicated that the weight of pituitary gland was reduced slightly below the control level but the ovarian and uterine weights of these animals were not different from those of the controls. The data on the males indicated that the pituitary gland and testes were slightly smaller in weight than those of the controls but the ventral prostate and seminal vesicles were not different significantly from those of the controls.
(4) The offsprings showing normal 4-days cyclicity in their vaginal smear had ovaries containing several generations of corpora lutea as well as follicles in various stages of development. However, the animals showing persistent cornification or no vaginal openings, could be divided into two types by the ovarian morphology ; one had ovaries consisting of only the large vesicular follicles with no corpora lutea and the other had ovaries consisting of both corpora lutea and vesicular follicles as observed in the normal cyclic rats.
(5) The anterior pituitary FSH contents of the female offsprings born from the mothers injected with TP were not different significantly from those of the control females, when determined by the modified method of Steelman and Pohley. The LH contents of the anterior pituitary gland of the female offsprings, measured by the ovarian ascorbic acid depletion method of Parlow, were reduced gradually according to the increase of TP dosages injected successively to the pregnant mothers, but it was not altered by the single injection of 5.0 or 10.0 mg of TP in late pregnancy.

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Measurement of luteinizing hormone (LH) in the urine has much significance in the diagnosis of menstrual disorders. Urinary LH assay has been previously carried out by troublesome, time-consuming bioassay. Recently, immunological method has become available for that purpose, since Wide and Gemzell (1961) reported LH-immunoassay using HCG antigen-antibody system which was based upon the cross-reaction between HCG and LH. It is the purpose of this report to demonstrate the cross-reaction between HCG and LH, to establish the immunological assay method of urinary LH and to investigate the relationship between immunological and biological activites of LH.
Anti-HCG sera were obtained from rabbits received 2 injections each of 1,000 IU of crude preparation of HCG (Primogonyl, HCG-P), freshly emulsified with complete Freund's adjuvant at two weeks interval. The injections were made in the footpads and i.d. After 3 weeks of rest, a series of booster injections with 1,000 IU of HCG-P without adjuvant were given i.p. nine times at every other day. Rabbits were bled 7 days after the last injection and the sera were kept. The antibody titers were in the range from 6,400 to 51,200 by tanned hemagglutination. Antiserum which had the highest antibody titer was used in this study.
This anti-HCG serum contained several antibodies which reacted not only with HCG but also with normal human serum (NHS) and child urine protein (CUP) by immunoelectrophoresis. In order to remove antibodies against proteins other than HCG, 1 ml of anti-HCG serum was absorbed with 30mg of lyophilized NHS and 20mg of CUP, thus the absorbed anti-HCG serum showed one precipitin band only to HCG. This absorbed serum also showed one precipitin band to human menopausal gonadotropin (Pergonal, HMG-P) which contained LH and FSH by agar gel diffusion and immunoelectrophoresis, and this band completely coalosced to the band wnich appeared against HCG.
This absorbed anti-HCG serum was examined for neutralization of LH and FSH. An assay method based on the rate of increase in the weight of the ventral prostates of hypophysectomized male rats (Greep) was used for LH assay and Steelman-Pohley's augmentation method was used for FSH assay. When 2 IU or 4 IU of LH and 2 IU or 4 IU of FSH were incubated with 0.002 ml of absorbed anti-HCG serum at 37°C for 60 min., biological activity of LH was completely neutralized but the FSH activity was not. From the above immunological and biological results, it could be concluded that LH had a similar antigen to HCG and LH was completely neutralized with anti-HCG serum.
The amount of LH in the urine is so small for immunoassay that concentration of urine using pervaporation, carbowax 6,000 and alcohol precipitation to 1 : 20 is necessary. The recovery of LH was almost complete. The components of medium to dissolve the standard HCG influenced the sensitivity of tanned hemagglutination inhibition reaction. The concentration from 1 to 1 : 20 of the child urine as the medium of HCG, showed 4 fold as sensitive as phosphate buffered saline, thus 1 : 2 concentrated child urine was used for the medium to dissolve the standard HCG or to dilute the test materials.
Ten women from 23 to 34 years old who had the normal menstrual cycle, were examined every day for BBT, and LH value in the first morning urine by immunoassay. Seven women who showed clearly biphasic BBT gave the peak of LH level, in the range from 80 IU to 320 IU HCG equivalent/L, from 15 to 13 days before next menstruation. These peaks coincided with the rise of BBT.
Three women among them were examined LH level in the urine by using immunological and biological methods simultaneously. The urine for each 48 hours during the cycle war collected, and COml of that was used for immunoassay and the rest was used for bioassay. The kaolin adsorption method by Bradbury-Matsushima was used to extract gonadotropin from the urine.

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