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The widespread clinical use of hemodialysis has prolonged the survival of a vast number of uremic patients, but it has also yielded some problems including renal osteodystrophy. Recently, it has become well known that the clinical use of active vitamin D metabolites is effective in many patients with renal osteodystrophy. However, there are many patients with bone diseases resistant to such treatment. Several lines of evidences implicate aluminum as one of the causal factors in the production of such diseases. We recently found 20 patients with bone diseases associated with the deposition of aluminum in front of active calcification in the bone. All of them were undergoing maintenance hemodialysis with softened water thrice weekly and taking aluminum containing antacids and 1 alpha-(OH)D3. The age of the patients ranged between 30 y.o. and 62 y.o. (46.5 +/- 9.0, mean +/- s.d.). All of them had severe bone pain and 8 of them had bone fractures. Bone X-ray, bone scintigraphy, serum Ca, P, ALP, serum aluminum and bone histology were examined. Based on the bone histology, they were classified into four types; inactive type (9), osteomalacia type (6), mild type (4) and mixed type (1). There were no significant differences among each group concerning serum values of Ca, P and aluminum. Serum value of ALP tended to be high in the osteomalacia type, and that of c-PTH was significantly low in the inactive type compared with the other types. Our finding suggest that aluminum associated bone disease is not so rare in Japan and show that the diagnosis of this disease should be made histologically and that clinical and blood chemical features are not reliable for the diagnosis of aluminum associated bone disease in hemodialysis patients.

Plasma concentrations of vitamin D3, 25-OH-D3, 24, 25(OH)2D3 and 1, 25(OH)2D3 were measured in patients with various diseases using the multiple assay system previously reported. In patients with hyperparathyroidism, the plasma levels of 1, 25(OH)2D3 tended to increase, while the levels of 24, 25(OH)2D3 tended to decrease in many cases. On the other hand, plasma 1, 25(OH)2D3 levels were low, while 24, 25(OH)2D3 levels were high in patients with hypoparathyroidism. No significant differences in the levels of plasma D3 derivatives among idiopathic-, postoperative- and pseudo- hypoparathyroidism were observed. In a majority of hemodialyzed patients with advanced renal failure who showed no overt bone changes on X-ray films, both the plasma 1, 25(OH)2D3 and 24, 25(OPH)2D3 levels were distributed from normal to very low. In a majority of patients with osteoporosis and hypoparathyroidism, plasma 1, 25(OH)2D3 levels rose quickly after the administration of 1 alpha-OH-D3. In hypophosphatemic vitamin D resistant rickets, however, the response to the relatively large amounts of 1 alpha-OH-D2 was poor, although the concentrations of plasma 24, 25(OH)2D3 were elevated by the 1 alpha-OH-D3 administration in most of these cases. The plasma 1, 25(OH)2D3 and 24, 25(OH)2D3 concentrations in patients with senile osteoporosis were distributed from lower to higher range than those of normal adults. There was a relatively good correlation between plasma levels of 25-OH-D3 and D3 only in cases with plasma D3 levels higher than 5 ng/ml. In addition, a hyperbolic regression curve was obtained between the plasma D3 and 25-OH-D3 ratio. These results may indicate that a possible negative feedback homeostatic mechanism exists between plasma levels of D3 and 25-OH-D3, but only with low plasma levels of D3.

In one case of untreated Hashimoto's disease, serum thyroxine (T4) value by radioimmunoassay (RIA) was significantly lower than that by competitive protein binding analysis (CPBA). The discrepancy was found to be due to the presence of antithyroxine autoantibody in the serum. This phenomenon was considered to be of practical importance in interpreting the T4 value by RIA in cases with autoimmune thyroid diseases. The patient was 59-year-old woman with a 30-year history of goiter. A diagnosis of Hashimoto's thyroiditis had been established by open biopsy of the thyroid ten years ago. The patient was judged to be euthyroid on the basis of clinical and laboratory evaluation (mean serum T4 by CPBA (Tetrasorb and Tetratab kit), 5.0 mug/100 ml; serum T3, 165 ng/100 ml; T3 resin uptake, 31.8%; and serum TSH, 2.0 muU/ml). TBG binding capacity was 24 mug/100 ml. Anti-thyroglobulin antibodies (anti-Tg), once positive ten years before, was negative at this time. But the mean T4 in the serum measured by T4 RIA and RIA-Mat T4 kit were 1.7 and 2.9 mug/100 ml, respectively. Recovery of the T4 added to the patient's serum evaluated by RIA-Mat T4 kit, was 71.2%, although the recovery using a control serum was 108%. Binding of 125I-T4 to the serum or fractions of the serum was studied by using polyethylene glycol (PEG) method, column chromatography, and double antibody precipitation. The results were as follows: 1) The binding of 125I-T4 to the patient's serum was detected by using RIA kit system without addition of anti-T4 serum. 2) On Sephadex G-200 chromatography of 125I-T4 incubated with the serum or the rabbit anti-T4 antibody in the presence of ANS, an early radioactive peak was observed by using the patient's serum as in the case of the anti-T4 antibody. When the serum after thermal inactivation of TBG, was incubated with 125I-T4, and was applied to the Sephadex G-200 column, a radioactive peak was observed in the area where 7S fraction was detected by protein peak. 3) The binding of 125I-T4 to the patient's IgG was 9.0% by using double antibody method when the binding to a control IgG was 0.5%. 4) The binding of 125I-T4 to IgG fractions was also proved by PEG method. 5) The binding of 125I-T4 was competitively inhibited by the addition of unlabeled T4. The affinity constant was 1.9 X 10(8) L/mol and its binding capacity was 0.8 mug/100 ml serum. From these data this T4 binding IgG was considered to be anti-T4 autoantibody. The cross reaction with T3 was approximately 8.3%. MIT and DIT did not displace labeled T4 when tested in amounts varying from 0.1 to 100 ng/assay. By using the paper electrophoresis, the binding of 125IT4 to the serum or IgG was not detectable. Therefore this method was considered unsuitable for detecting such anti-T4 antibody. As we couldn't find any significant binding of 125I-T4 to sera in 37 other patients with Hashimoto's disease by using the PEG method, the incidence of this phenomenon was considered to be low...

A simple and precise method has been developed for the determination of 25-hydroxyvitamin D in 1 ml of human plasma. The method consists of methanol/chloroform extraction, purification by high pressure liquid chromatography and a competitive protein binding assay using vitamin D deficient rat serum. The ethanol extract from vitamin D deficient chick serum was added to the sample before CPBA to eliminate the non-specific interference in the CPBA system as a vitamin D free serum extract. The assay was sensitive to 0.72 ng/ml of plasma. Satisfactory results were obtained in the dilution and recovery tests. The coefficients of variation were 5.8 approximately 9.1% for the within-assay, and 7.4 approximately 10.3% for the between-assay. Plasma concentrations of 25-hydroxyvitamin D in 46 samples of normal human plasma were 21 +/- 10.5 ng/ml (mean +/- SD), and the seasonal variation was demonstrated. Plasma levels for 25-hydroxyvitamin D were high in patients receiving vitamin D2 and low in patients suffering from liver cirrhosis.

The existence of Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C), the effect of gastrin on phospholipid metabolism and guanylate cyclase activity were investigated to elucidate the cellular mechanism of action of gastrin on the corporal mucosa of the canine stomach. Protein kinase activity was determined by measuring the incorporation of [32P] into calf thymus H1-histone from [32P]-ATP. One unit of protein kinase was defined as the amount of enzyme which incorporated 1 pmol of phosphate from ATP into H1-histone. Protein kinase C was found in 100,000xg supernatant of homogenate fractionated by a DEAE-cellulose column chromatography. Characteristics of further purified protein kinase C, such as dependency on divalent cations and phospholipids, were in agreement with those of previously reported protein kinase C in other tissues. Furthermore, the gastric corporal mucosa was found to contain protein kinase C in large quantities. The specific activity of protein kinase C was 26,000 units/mg protein. The phospholipid metabolism was evaluated by the incorporation of [14C]-glycerol-3-phosphate and the change of the radioactivity of [32P] in individual phospholipids. Each phospholipid was extracted from the gastric corporal mucosa and isolated by thin layer chromatography. Guanylate cyclase activity was determined by measuring the cGMP produced, using radioimmunoassay. Gastrin significantly increased the incorporation of [14C]-glycerol-3-phosphate into phosphatidylethanolamine in the presence of acetylcholine (Ach). Ach increased the uptake of the tracer into phosphatidylinositol significantly, and the increase was enhanced by the simultaneous addition of gastrin. In the experiments with [32P]-labeled phospholipids, gastrin increased the incorporation of [32P] into phosphatidylethanolamine significantly. The significant increase of the radioactivity in phosphatidylinositol by Ach failed to be enhanced by gastrin, but that of phosphatidylethanolamine by Ach was enhanced by gastrin. No stimulation of guanylate cyclase activity by gastrin was detected in the dispersed gastric corporal mucosal cells. These results indicate that gastric corporal mucosa was one of the most abundant tissues in which protein kinase C was contained, when compared with various mammalian tissues previously reported by Minakuchi, Nishizuka, et al. Nishizuka et al, recently proposed the novel hypothesis that phosphatidylinositol turnover activated by cAMP-independent agonists will be essentially required to activate protein kinase C. Our results suggest that gastrin can provoke phospholipids turnover including phosphatidylinositol turnover in gastric corporal mucosa. Therefore, our data indicate the possibility that the protein kinase C system plays an important role in the cellular mechanism of action of gastrin on gastric corporal mucosa.

The prognostic significance of both the triiodothyronine (T3) suppression test and the detectability of thyroid stimulating immunoglobulins in patients with Graves' disease who had been treated with antithyroid drugs was evaluated. Eighty-three patients underwent a T3 suppression test after having been euthyroid for at least 6 months. In 33 patients, the human thyroid stimulator (HTS) was assayed by measuring cyclic AMP increase in cultured thyroid adenoma cells, and TSH-binding inhibitor immunoglobulins (TBII) were measured by using the radioreceptor assay of TSH. Among 43 patients who had discontinued the drug treatment, 37 patients were under observation for 6-42 months. When a fall in 30-minute thyroid 99mTcO4- uptake of 50% or more after T3 administration was defined as positive suppression, the relapse rate was 30% in non-suppressive cases and 26% in suppressive cases. The relapse rate was lower in cases whose pre-suppression uptake was less than 3.0% (3 out of 17 patients) or in cases whose uptake after T3 administration was less than 0.8% (1 out of 9 patients). Of 15 patients with negative suppression, 5 (33.3%) were positive in HTS and 4 (26.7%) were positive in TBII. On the other hand, two (11.1%) each of 18 patients with positive suppression were positive in HTS and TBII respectively. Neither HTS nor TBII had been detected at the cessation of therapy in any of the 12 patients who remained euthyroid during the follow-up period. On the other hand, four (44.4%) out of 9 patients who relapsed had been positive in either HTS of TBII. Thus the Graves' disease specific immunoglobulins were found to be significantly associated with the relapse of the disease (p less than 0.05). The above data indicates that regardless of the suppressibility of 99mTcO4- uptake after T3 administration, the rate of recurrence is high when the uptake after T3 is more than 0.9% and/or when either HTS or TBII are positive.

Radioisotopes have been widely used in both in vitro and in vivo studies of endocrine organs. In this lecture, results of our recent in vitro studies on calcium regulating hormones, thyroid-stimulating hormone, and gastro-intestinal hormones are reviewed. 1) Clinical evaluation of measurement of calcium regulating hormones such as PTH, calcitonin and vitamin D metabolites was demonstrated. Furthermore, problems of those assays were also discussed. Especially, simpler methods which measure intact PTH or vitamin D metabolites are to be developed. PTH-like factor, which should play the major role in hypercalcemia of malignancy, was assayed biologically. After determination of amino-acid sequence of this protein, clinical measurement should reveal mechanism of hypercalcemia of malignancy. 2) Studies on TSH-receptor antibodies by using radioreceptor assay. TSH-binding inhibitor immunoglobulins (TBII) were detected not only in 93% of untreated patients with Graves' disease, but also in 21% of patients with primary myxedema. In contrast to the thyroid-stimulating nature of TBII in Graves' patients, TBII in hypothyroid patients were disclosed to be blocking in their nature. Clinical and laboratory findings supported pathogenetic role of the blocking antibodies in the latter condition. 3) Mutual regulation between CCK and muscarinic receptors on dispersed pancreatic acini. CCK and carbachol in their higher concentrations regulated muscarinic receptor and CCK receptor, respectively. The mode of regulation of both receptors was disappearance of their high affinity binding site. TPA, an activator of protein kinase C, modulated both receptors in the same manner as CCK or carbachol. These effects of CCK and carbachol on receptors were well compatible to the restriction of carbachol or CCK induced amylase secretion by CCK or carbachol. These in vitro studies, in association with the results of in vivo studies, contribute to the developments of nuclear endocrinology.