Hypothyroidism has been shown to be accompanied by hyperlipoproteinemia. To examine the pathogenesis of hyperlipidemia in hypothyroidism, plasma cholesterol, triglyceride, lipoprotein electrophoresis on agarose gel and PHLA were measured in 8 patients with hypothyroidism before and during thyroid hormone replacement therapy. Lipoprotein lipase was measured as protamine-inactivated lipase activity; hepatic triglyceride lipase activity was measured as protamine-resistant lipase activity by the method of Krauss.
Lipoprotein phenotyping revealed type IIa hyperlipoproteinemia in 3 patients, type III in 4 patients and type IV in 1 patient.
The mean±S.E.M. of fasting plasma cholesterol and triglyceride were 258.8±25.2 and 191.5±51.1 mg/dl in the pretreatment period and were 199.9±19.9 and 131.1±30.4 mg/dl on thyroid hormone replacement, respectively.
Hepatic triglyceride lipase was lower in the patients with hypothyroidism (4.51±0.65k moles FFA/ml/h) than in the controls (13.80±1.85, p<0.001) and increased (10.20±1.46, p<0.01) with treatment to levels comparable to those of the controls. However, lipoprotein lipase in the patients with hypothyroidism (2.76±0.43μ moles FFA/ml/h) was not different from that of the controls (3.56±0.76) and did not increase (3.33±0.69) significantly with treatment.
The effects of a high carbohydrate diet and a high fat diet on lipoprotein metabolism were studied in 4 patients with hypothyroidism. Changing from a high carbohydrate diet to a high fat diet produced a mean decrease of plasma triglyceride (98.5 mg/dl) and a change of lipoprotein phenotype from type III to type IIa in 1 patient. Electrophoretograms in the other 3 patients (type IIa, 2 patients; type III, 1 patient) did not change.
Two euthyroid volunteers were administered 300 μg of 1-thyroxine daily for two weeks and showed a significant increase in hepatic triglyceride lipase compared with their normal pretreatment levels.
These results suggest that low hepatic triglyceride lipase is responsible for an abnormal VLDL conversion to LDL in hypothyroidism as Krauss et al. reported. Moreover, the nutritional state in addition to the low PHLA may produce either type II, type III or type IV hyperlipoproteinemia in hypothyroidism.

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In order to investigate plasma bioactive PTH, we tried to assay the N-terminal portion of PTH by RIA. The antiserum to PTH was prepared by immunizing rabbits with a bovine 1-34 PTH conjugate BSA. A preparation of labeled PTH was radioiodinated by the chloramine-T or lactoperoxidase method. Labeled PTH was purified by means of adsorption by Quso G-32 powder or a sephadex G-50. The separation of the free and bound labeled hormone was performed by the dextran-coated charcoal method. The assay was carried out as follows : 0.2 ml diluted buffer (0.05M, pH 8.6, veronal buffer), 0.1 ml standard PTH or sample to be tested, and 0.1 ml anti-PTH serum were mixed. After the first incubation at 4°C for 4 days, 0.1 ml labeled PTH were added. After a second incubation at 4°C for 12 hours, the assay tubes were centrifuged at 2,000 rpm for 30 min and the precipitates were counted. Various hypothalamic, pituitary and thyroid hormones did not interfere with the RIA for PTH. A dose response curve was obtained in a range from 100 pg to 5,000 pg per ml of standard PTH in this assay system. The serum immunoreactive PTH in healthy subjects values less than 290 pg per ml.

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In order to establish radioimmunoassay (RIA) systems for the determination of TSH subunits were carried out.
Standard TSH supplied from MRC and standard TSH subunits from Calbiochem were identified to be highly purified by gel filtrations through a Sephadex G-100 column.
Antibodies of TSH subunits supplied from Calbiochem bound to standard TSH subunits but not to other pituitary hormones except for TSH.
Cross reactivity of and TSH-a antibody to standard TSH was 0.000082, and that of TSH-13 to standard TSH was 0.000011. As the standard curve of each subunit and the displacement curve by TSH were parallel in each assay system, serum levels of TSH subunits were computed by the subtraction of the amount of TSH subunits which was overestimated by the cross reaction with native TSH in this assay system.
Cross reactivity of and TSH-α antibody to TSH-β was 0.001, and that of and TSH-β antibody to TSH-β was 0.002. But in clinical studies, one TSH subunit was not greatly affected by the determination of another TSH subunit.
Utilizing these standard TSH subunits, antibodies of TSH subunits and ¹²⁵I-labelled TSH subunits obtained by the chloramine-T method, the RIA systems for measurement of serum TSH subunits levels without an extraction procedure were developed.
In each RIA system of TSH subunit, a good dose response curve was observed in the range from 0.2 to 50 ng/ml of TSH subunits. Recovery and reproducibility of each RIA system were satisfactory.
The serum levels of TSH subunits in 13 normal subjects, 12 patients with primary hypothyroidism and 7 patients with hyperthyroidism were determined by the RIA of TSH subunits.
In 13 normal subjects, serum levels of TSH-α varied from an undetectable level (U, D. : <0.2 ng/ml) to 0.3 ng/ml, and those of TSH-β varied from an undetectable level (U.D. : <0.2 ng/ml) to 0.8 ng/ml.
In 12 patients with primary hypothyroidism, serum levels of TSH-α varied from U.D. to 4.9 ng/ml, and those of TSH-β varied from U.D. to 13.6 ng/ml.
In 7 patients with hyperthyroidism, serum levels of TSH-α varied from U.D. to 4.3 ng/ml, and those of TSH-13 were ranged U.D..
From the above data, it is suggested that the direct RIA of serum TSH subunits is a useful tool for studying the roles of TSH subunits in peripheral blood.

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In order to investigate the physiological and pathophysiological roles of TSH subunits, changes of serum TSH, its subunits, and T₃ and T₄ levels before and after the intravenous administration of 500μg synthetic TRH were examined in 13 normal subjects and 125 patients with endocrinopathy.
Serum levels of TSH, its subunits, and T₃ and T₄ were determined directly by each specific radioimmunoassay.
The responsiveness of serum TSH to TRH administration was the same as that in our previous report and was observed to be regulated by serum T₃ and T₄.
In 13 normal subjects, serum levels of TSH subunits increased differently from one another after the TRH administration. Basal and peak levels of TSH-a varied from an undetectable level (U.D. : <0.2 ng/ml) to 0.3 ng/ml, and from 0.2 ng/ml to 1.8 ng/ml respectively. Basal and peak levels of TSH-β varied from an undetectable level (U.D. : <0.2 ng/ml) to 0.8 ng/ml, and from 1.4 ng/ml to 17.5 ng/ml respectively.
In 12 patients with primary hypothyroidism, basal and peak levels of TSH-α varied from U.D. to 4.9 ng/ml, and from 2.6 ng/ml to 5.0 ng/ml respectively. Basal and peak levels of TSH-β varied from U.D. to 13.6 ng/ml, and from 3.3 ng/ml to 48.7 ng/ml respectively.
In 3 patients with secondary hypothyroidism, basal and peak levels of TSH-α varied from U.D. to 0.4 ng/ml, and from 0.4 ng/ml to 0.9 ng/ml respectively. Levels of TSH-β remained at U.D. before and after the TRH administration.
In one patient with tertiary hypothyroidism, basal and peak levels of TSH-α ranged at U.D. and 0.7 ng/ml. Basal and peak levels of TSH-β were ranged at U.D. and 0.8 ng/ml.
In 7 patients with hyperthyroidism, basal and peak levels of TSH-α varied from U.D. to 4.3 ng/ml, and from 0.6 ng/ml to 6.9 ng/ml respectively. Levels of TSH-β remained at U.D. before and after the TRH administration.
In 6 patients with euthyroid Graves' disease, basal and peak levels of TSH-α varied from U.D. to 1.5 ng/ml, and from 0.2 ng/ml to 2.0 ng/ml respectively. Basal and peak levels of TSH-β varied from U.D. to 2.0 ng/ml, and from 0.9 ng/ml to 5.5 ng/ml respectively.
In 7 euthyroid hypothalamo-pituitary disorder, 7 anorexia nervosa, 3 Cushing's disease, one diabetes insipidus, 7 acromegaly and 2 Turner syndrome, levels of TSH subunits remained at around U.D. before and after the TRH administration.
A positive correlation was observed between basal or peak levels of TSH-β and those of TSH, and a negative correlation was observed between basal or peak levels of TSH-β and those of T₃ or T₄, while the correlation was not observed between basal or peak levels of TSH-α and those of TSH, T₃ or T₄.
From the above data, it is suggested that :
1. TSH subunits exist in human serum and are released from pituitary to peripheral blood after a TRH administration.
2. TSH-α does not respond significantly after a TRH administration.
3. TSH-β responds significantly after a TRH administration, and its response is regulated by T₃ and T₄.
4. In patients with secondary hypothyroidism and hyperthyroidism, the decrease of TSH reserve is accompanied by the decrease of TSH-β synthesis.
5. TSH-β has some pathophysiological roles in some cases of euthyroid Graves' disease.

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Although there is general agreement on the distribution of catecholamines in the brain, controversies exist with regard to the results of investigations on their physiological roles in the regulation of gonadotropin secretion. In most of the studies, catecholamines were injected into the third ventricle, and the effects of catecholamines on gonadotropin secretion were examined in in vitro experiments. Since these experiments did not allow any precise localization of the sites of action of catecholamines, the present study was undertaken in order to determine where noradrenaline (NA), dopamine (DA) and acetylcholine (ACH) are involved in the regulation of gonadotropin secretion in the limbic-preoptic-hypothalamic system of the female rat.
Wistar female rats which were ovariectomized 2 or 3 weeks earlier and animals showing two consecutive regular 4 day estrous cycles were used for the experiments. Blood samples were taken by heart puncture under light ether anesthesia, and serum LH and FSH were measured by radioimmunoassay.
First, in order to examine the involvement of NA, DA and ACH in the regulation of progesterone-induced gonadotropin release, receptor blockers of NA, DA or ACH, phenoxybenzamine (20 mg/kg body weight, i.p., in 50% ethanol), pimozide (1 mg/kg body weight, i.p., in 0.1 M tartaric acid) or atropine (700 mg/kg body weight, s.c., in saline), respectively, were administered to estrogen (20 μg of estradiol benzoate) and progesterone (2 mg) -treated ovariectomized rats. Injections of 2 mg of progesterone into ovariectomized estrogenprimed rats significantly increased serum LH and FSH concentrations 3, 5 and 8 hr later. Phenoxybenzamine, pimozide or atropine prevented the progesterone-induced LH and FSH release.
Secondly, rats were pretreated with three blockers to determine whether NA, DA and ACH participate in the regulation of gonadotropin release in response to electrochemical stimulation of the medial preoptic area (MPO). Electrochemical stimulation of the MPO (100 μA for 60 sec) in ovariectomized estrogen-primed rats produced a dramatic rise in serum LH with a peak 1.5 hr later and in serum FSH with a peak 4.0 hr later. None of the blockers eliminated this gonadotropin release, although pimozide or atropine reduced serum LH concentrations at 4.0 hr after stimulation. In pentobarbital (35 mg/kg body weight) blocked proestrous rats, electrochemical stimulation (50 i/A for 30 sec) of the MPO induced a marked LH release 1.5 hr later resulting in occurrence of ovulation. The administration of phenoxybenzamine did not change the LH release following MPO stimulation and induction of ovulation.
Thirdly, the sites of action of NA, DA and ACH with respect to LH release in the limbic-preoptic system were determined by intracerebral implantation in ovariectomized estrogen-primed rats. DA or ACH, when implanted unilaterally into the MPO, induced a significant increase in serum LH 5 hr later, whereas NA decreased LH levels. Implantations of NA or ACH into the nucleus of the stria terminalis or the medial amygdala increased serum LH, although the effect of NA into the latter was not statistically significant. Only implantations of NA among the three substances into the lateral septum induced LH release.
The results mentioned above showed that NA, DA and ACH all play, possibly as neurotransmitters, stimulatory roles in the regulation of progesterone-induced gonadotropin release in ovariectomized estrogen-primed rats. Furthermore, it would seem most reasonable to propose that none of the adrenergic, dopaminergic and cholinergic synapses are involved in mediating the effect of electrochemical stimulation of the MPO to the medial basal hypothalamus, and that electrochemical stimulation activates directly LH-RH neurons or LH-RH containing neural elements in the MPO to induce discharge of LH-RH from the axon terminals in the median eminence into portal vessels.

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Although it is well known that the limbic-preoptic-hypothalamic system regulates gonadotropin secretion from the pituitary gland, there have been few studies on the involvement of the midbrain in the control of ovulation. Therefore, in the present study, an investigation was made to determine which portion of the midbrain is involved in the control of ovulation and the proestrous surge of gonadotropins and prolactin (PRL) by placing various transections in the rat brain, and their effects were evaluated by comparison with those under other hormonal conditions.
Wistar male rats and females showing two consecutive regular 4 day estrous cycles were used for the experiments. Blood samples were taken through an indwelling atrial cannula without anesthesia, and serum LH, FSH and PRL were measured by radioimmunoassay. Bilateral lesions in the ventral tegmental area (VTA) were made by applying 2.5 mA of DC current for 20 sec through a platinum electrode. In order to cut the neural element in the three different portions of the midbrain, a small knife of bayonet shape was used.
The following results were obtained :
(I) Bilateral lesions in the VTA made in the morning of proestrus, which destroyed more than 50% of the bilateral sides of the VTA, blocked ovulation in 5 out of 10 animals (P<0.05), delayed the peak of the proestrous surge of FSH and PRL by 2 hrs, and significantly lowered serum LH, FSH and PRL levels at 1800 hr of proestrus. Similarly, incomplete lesions in the VTA partially inhibited the gonadotropin surge whereas they failed to block ovulation.
(II) Midline transections placed in the morning of proestrus, which cut the neural tissue in the ventromedial portion of the midbrain (VMT), were effective in blocking ovulation in 9 out of 10 animals, and in the ovulation-blocked rats, the proestrous surge of LH, FSH and PRL was completely eliminated. Midline transections, which cut the neural tissue in the dorsal portion of the midbrain, neither blocked ovulation nor affected serum gonadotropins and PRL levels at any time. Bilateral transections (LT), which interrupted the neural tissue located laterally to that transected by VMT, failed to block ovulation. Following this type of transection, rather significantly higher serum LH levels were observed at 1400, 1600 and 2000 hr of proestrus as compared to those of the sham transected control group.
(III) VMT made in the evening of proestrus had no effect on the secondary rise in serum FSH which occurred at midnight between proestrus and estrus. Furthermore, VMT did not affect the postcastration rise in serum LH and FSH in males which was observed at 24 hr after orchidectomy. Although some ovariectomized or orchidectomized animals with VMT showed pulsatile. LH release with decreased frequency and high amplitude, complete disappearance of pulsatile release could be observed in only 1 out of 5 animals.
The findings described above showed that the ventromedial portion of the midbrain contains the neural structure indispensable for the occurrence of the proestrous surge of gonadotropins and PRL. The specificity of the effects of VMT was demonstrated by the failure of VMT to have any effects on the other conditions of gonadotropin secretion. In view of the fact that LT, which is considered to interrupt a greater portion of the ventral noradrenergic pathway projecting to the preoptic area and the hypothalamus than does VMT, was without effect in blocking ovulation, it is possible that the essential neural element is not noradrenergic. Furthermore, the neural mechanism for the secondary rise in serum FSH was shown to be different from that for the proestrous surge of FSH which is associated with LH release, and it was also suggested that the lateral area to the essential neural structure for ovulation contains an inhibitory mechanism for LH release.

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Basal plasma levels and urinary excretion of adenosine 3', 5'-monophosphate and guanosine 3', 5'-monophosphate were determined in patients with primary hyperparathyroidism, idiopathic, surgical and pseudohypoparathyroidism and in normal controls in order to elucidate whether or not they provide good indices of parathyroid activity. The effects of the parathyroid hormone (PTH), calcium and calcitonin (CT) on plasma and urinary c-AMP and c-GMP were studied in patients with hyperparathyroidism and hypoparathyroidism and in normal controls.
I) C-AMP and c-GMP were measured in urine collected from 8 : 00 a.m. to 10 : 00 a.m. and in plasma obtained at 9 : 00 a.m. in 9 patients with primary hyperparathyroidism, in 24 patients with hypoparathyroidism and in 9 normal controls.
II) 200 units of bovine PTH (Eli-Lilly) were injected intravenously with saline at 10 : 00 a.m. Urine was collected at 30, 60,120 and 180 min., and blood was obtained at 30, 60 and 120 min. after the PTH administration.
III) Calcium (Ca 15 mg/kg) was infused intravenously with 500 ml of saline for 4 hours (9 : 00 a.m. to 1 : 00 p.m.). Urine was collected, and blood was obtained at every 1 hour until 1 : 00 p.m.
IV) 40 MRCU. of calcitonin was injected intramuscularly, urine was collected and blood was obtained every 1 hour until 1 : 00 p.m
Urine and plasma c-AMP and c-GMP were measured by radioimmunoassay using YAMASA c-AMP and c-GMP assay kits. Serum PTH was measured by radioimmunoassay using an antibody to bovine PTH.
The results were as follows : 1) The basal excretion of urinary c-AMP was significantly higher in 9 patients with primary hyperparathyroidism and significantly lower in 24 patients with hypoparathyroidism than it was in the normal controls. There were positive correlations between these basal excretions and serum Ca or PTH levels.
The plasma c-AMP levels were higher in patients with primary hyperparathyroidism than they were in the normal controls, but those with hypoparathyroidism were not so different from the normal controls. On the other hand, plasma and urinary c-GMP did not show any significant differences between the normal controls and patients with hyper or hypoparathyroidism. 2) The responsiveness of urinary c-AMP to PTH was higher in the patients with hypoparathyroidism and lower in the patients with primary hyperparathyroidism than it was in the normal controls. In the patients with pseudohypoparathyroidism, however, only a small increase was observed. The responsiveness of plasma c-AMP showed the same tendency though much less than urinary c-AMP. Urinary c-GMP showed about a 50% increase in the urine collected the first 1 hour after the PTH administration, but there was no apparent difference between the normal controls and the patients with hyper- or hypoparathyroidism. 3) The urinary excretion of c-AMP decreased by calcium infusion for 4 hours in patients with primary hyperparathyroidism because of the suppression of endogenous PTH secretion. On the other hand, urinary c-GMP increased in 5 out of 7 patients. In a case of pseudohypoparathyroidism, c-AMP excretion increased markedly after the calcium infusion. 4) Urinary c-AMP excretion increased 55% in 3 normal controls and 39% in 5 patients with primary hyperparathyroidism by 40U of CT. This increase may have resulted from the direct effect of calcitonin. 5) The urinary excretion of c-AMP decreased immediately after parathyroidectomy in patients with primary hyperparathyroidism.
From these findings, it is suggested that c-AMP in plasma and urine provides a good index of parathyroid activity, but c-GMP does not.

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It has been reported that mental retardation due to congenital hypothyroidism can be prevented by early detection and early adequate replacement therapy. We have developed a radioimmunoassay for TSH using the dried blood spot and have started screening for newborn congenital hypothyroidism using a part of sample of the inborn metabolic error screening.
(1) The dried blood spot TSH of 61,000 newborn infants was assayed in the first half of our screening and that of 74,505 newborn infants was assayed in the latter half of our screening. As a result, although we were not able to detect any cases in the first screening, we were able to detect 9 cases of congenital hypothyroidism in the latter screening. From the results obtained through our investigation of the thyroid function of these 9 infants, we confirmed that mild hypothyroidism can be better detected by the screening of TSH.
(2) As to the program of the screening, we chose from the latter half of our screening all the samples in which TSH concentrations contained above 3 percent of each assay and were remeasured on the next assay.
(3) As we confirmed that the sensitivity of measurement was increased at very low concentrations, when the volume of antibodies, radioisotopes and eluates used for each assay were decreased, we measured TSH successfully using two 3 mm discs.
(4) As we can perform very simple screening by the 3 mm disc method, we are changing the screening method from that with 10 mm disc to one with two 3 mm discs.
We intend to extend our screening, and will make every effort to prevent mental retardation due to congenital hypothyroidism.

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