Dose effects of oral estradiol on bone mineral density in Japanese women with osteoporosis.

OBJECTIVES: This 2-year study compared 0.5 and 1.0 mg oral estradiol (E(2)), with or without levonorgestrel (LNG), for the treatment of postmenopausal osteoporosis in Japanese women. METHODS: Japanese women with osteoporosis after natural menopause or bilateral oophorectomy were randomized to receive E(2) 0.5 or 1.0 mg/day with LNG 40 microg as required, or placebo, for 52 weeks. Women treated with E(2) in the first year continued therapy at the same doses in the second year. Efficacy, safety and pharmacokinetics were assessed. RESULTS: There were 73 women randomized to E(2) 0.5 mg, 157 to E(2) 1.0 mg and 79 to placebo. Lumbar bone mineral density at 52 weeks increased significantly more with E(2) 1.0 mg (p < 0.001) and 0.5 mg (p < 0.001) than with placebo (no change). After 2 years, a 10% increase in bone mineral density with E(2) 1.0 mg was significantly greater than with E(2) 0.5 mg (8%; p = 0.008). E(2) was associated with an acceptable safety and tolerability profile, with slightly more adverse events with E(2) 1.0 than 0.5 mg. Serum E(2) concentration increased in a dose-dependent manner. CONCLUSION: This study showed that E(2), at both 1.0 mg and 0.5 mg doses, was effective in increasing bone mineral density with an acceptable safety and tolerability profile in Japanese postmenopausal women with osteoporosis but that the bone mineral density response was higher with the 1.0 mg dose.

Neurophysin biosynthesis in vitro in oat cell carcinoma of the lung with ectopic vasopressin production.

The incorporation of labeled compounds into neurophysins of a transplantable human oat cell carcinoma of the lung with ectopic vasopressin production was studied in vitro. Neurophysins in cell extracts and in incubation media were isolated by immunoprecipitation and analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. When cells were incubated with L-[35S]cysteine for 12 h, SDS-polyacrylamide gel electrophoresis of the immunoprecipitates from cell extract and medium resolved two forms of neurophysins with apparent molecular mass of 10,000 (10K) and 20,000 (20K). Both forms of [35S]-neurophysins were completely displaced from the immunoprecipitates by excess human neurophysin. Incubation of cells with L-[35S]cysteine and D-[3H]-glucosamine hydrochloride revealed that glucosamine was incorporated into the 20K neurophysin region, but not into 10K species. To observe the kinetics of labeling of the two forms of neurophysins, cells were incubated with L[35S]cysteine for varying periods of time. After short labeling periods, most of the radioactivity resided in 20K species, which plateaued after 1 h, whereas 10K neurophysin progressively increased in its height. When cells were chased with unlabeled cysteine after the exposure to a short pulse of labeling, 20K neurophysin peak gradually decreased with an apparent initial half-life of 1 h. In contrast, the label in 10K neurophysin steadily increased, which exceeded the former by 3 h of chase. Analysis of 20K neurophysin in cell extract by isoelectric focusing on polyacrylamide gel demonstrated that it was principally composed of a protein with an apparent isoelectric point (pI) of 5.7. These results suggest that neurophysin is synthesized in ectopic vasopressin-producing tumors by post-translational processing from a glycosylated proneurophysin with an apparent molecular mass of 20,000 daltons and a pI of 5.7.

Effect of captopril on glucose concentration. Possible role of augmented postprandial forearm blood flow.

The goal of this study was to evaluate the effects of captopril on plasma glucose concentration. The daily profiles of the plasma glucose levels were determined in 12 non-insulin-dependent diabetic normotensive subjects, treated with or without captopril at a dose of 25 mg 3 times/day. Forearm blood flow was also measured by strain-gauge plethysmography. Administration of captopril improved the daily profile of the plasma glucose level. Postprandial forearm blood flow was also augmented 2 h after a meal. These results suggest that angiotensin-converting enzyme inhibitors may improve glucose metabolism in diabetic subjects, possibly through enhancement of blood flow to skeletal muscle.

A new method of bone tissue measurement based upon light scattering.

In recent years, time-resolved spectroscopy systems using near infrared pulsed laser have been applied to develop optical computed tomography. We applied this technique to measure the optical properties of osseous tissues. First, we gradually demineralized 10 mm blocks of bovine trabecular bone with EDTA, maintaining the absorption characteristics and structure but varying the hydroxyapatite content, thus creating specimens differing only in light scattering properties. We used computer densitograms to assess light penetration, and analyzed the correlation with bone mineral density (BMD) as with dual-energy X-ray absorptiometry scans. The light penetration increased with decreasing BMD. Second, using the above-mentioned pulsed laser time-resolved spectroscopy system, we investigated the correlation between the BMD and the time response waveforms of 10-mm blocks of bovine cortical bone, trabecular bone, and surrounding tissue as well as human trabecular bone. The human lumbar vertebral bone also displayed an inverse correlation between BMD and maximum light penetration and a positive correlation between BMD and peak time delay. This is the first demonstration of a correlation between BMD and light scattering properties showing that BMD can indeed be measured with light. Our results show the possibility of obtaining information on internal bone structure and composition in vivo through assessment of the waveforms obtained by a time-resolution system in the near infrared region.

Serum dehydroepiandrosterone sulfate in Cushing's syndrome.

Serum concentrations of dehydroepiandrosterone sulfate (DHEA-S) were measured in patients with hyperadrenocorticism. When compared to normal subjects of corresponding age, serum DHEA-S levels were normal or elevated in 37 patients with Cushing's disease. In contrast, DHEA-S levels were significantly lower than those of normal subjects in all 28 patients with hyperadrenocorticism due to benign adrenocortical adenoma, suggesting that ACTH is the major determinant of DHEA-S secretion and that determination of serum DHEA-S concentrations is useful in the biochemical differential diagnosis of the etiology of Cushing's syndrome. In six patients with adrenocortical adenoma, the recovery of suppressed DHEA-S secretion after removal of the adrenal gland affected by a tumor was studied. Serum cortisol levels normalized by the end of the second year after unilateral adrenalectomy, while DHEA-S levels remained low for at least 2 succeeding yr. The results suggest that deficient ACTH secretion may result in a greater and longer lasting loss in the ability of the adrenal cortex to secrete androgens than in the ability to secrete cortisol.

Hyperreninemia due to increased renal renin synthesis in BioBreeding Worcester rats.

It is well known that diabetes mellitus is often associated with hypertension. We previously reported the unresponsiveness of renin release to volume depletion with impaired renal prostaglandin E2 synthesis in rats with streptozotocin-induced diabetes. However, we have found that BioBreeding Worcester rats, spontaneously susceptible to diabetes mellitus either before or after the onset of diabetes, showed a pronounced fourfold to ninefold increase in plasma renin activity in comparison with control Wistar rats. Furthermore, these rats developed mild hypertension as high as 134 mm Hg after the age of 90 days. The hyperreninemia responded to 1-week sodium loading or restriction; the blood pressure increased during sodium loading. Oral administration of captopril (30 mg/kg) for 1 week resulted in a large blood pressure decrease (-47.1 +/- 5.9 mm Hg, n = 10) in comparison with controls (-17.0 +/- 4.7 mm Hg, n = 12). Vascular response to angiotensin II was also attenuated. Plasma angiotensin II levels were 5.7-fold higher and associated with a 1.5-fold increase of plasma aldosterone concentration compared with control rats, whereas angiotensinogen-plasma concentrations were lower than in control rats. The renal renin content determined enzymatically or histochemically was more enhanced in BioBreeding Worcester rats than in control rats, but the renal renin messenger RNA levels did not differ. These results suggest that the strain-specific hyperreninemia in BioBreeding Worcester rats might be due to posttranscriptional abnormalities of renal renin synthesis. Further work is needed to elucidate the specific mechanism or mechanisms responsible.

Serum dehydroepiandrosterone sulfate concentrations in secondary adrenal insufficiency.

Serum dehydroepiandrosterone sulfate (DHEA-S) concentrations were studied in 84 untreated patients with secondary adrenal insufficiency. Compared to values in normal subjects of corresponding age, DHEA-S levels were decreased in 80 patients. The decrease was unrelated to the cause of the secondary adrenal insufficiency or the serum PRL level. Serum cortisol concentrations, on the other hand, were low in 71 patients and low normal in the remaining 13. Serum DHEA-S levels were decreased in 11 of these 13 patients. The frequency of decreased serum DHEA-S levels in patients with secondary adrenal insufficiency was significantly higher than that of decreased cortisol levels. These results suggest that decreased serum DHEA-S levels reflect deficient ACTH secretion in secondary adrenal insufficiency and that simultaneous determination of serum DHEA-S and cortisol levels is useful in the diagnosis of this pathological state.

Plasma levels of atrial natriuretic hormone in Cushing's syndrome.

To examine a possible role for atrial natriuretic hormone (ANH) in the water and electrolyte disturbances associated with hypercortisolism, plasma ANH levels were measured in 18 patients with endogenous Cushing's syndrome. Nine patients had elevated plasma ANH levels compared to normal subjects. The mean plasma ANH concentration [72.5 +/- 13.0 (+/- SE) pg/mL (23.5 +/- 4.2 pmol/L)] in the Cushing's syndrome patients was significantly higher than that in 40 normal subjects [37.6 +/- 1.9 pg/mL (12.2 +/- 0.62 pmol/L)]. A significant positive correlation was found between plasma ANH and cortisol levels in individual patients. There were no significant correlations, on the other hand, between plasma ANH concentrations and PRA, plasma aldosterone levels, or mean blood pressure. After treatment, plasma ANH concentrations decreased in all 6 patients who had elevated plasma ANH levels preoperatively. In 1 patient with Cushing's disease, plasma ANH levels changed in parallel with plasma cortisol concentrations during o,p'DDD treatment. Fifteen patients who were receiving long term synthetic glucocorticoid therapy for the treatment of miscellaneous diseases had a significantly higher mean plasma ANH level [50.2 +/- 4.0 (+/- SE) pg/mL (16.3 +/- 1.3 pmol/L)] than that in normal subjects. These results suggest that plasma ANH levels are elevated in a substantial number of patients with Cushing's syndrome due to either a direct stimulatory effect of glucocorticoid on atrial ANH secretion or, alternatively, intravascular volume expansion resulting from excessive cortisol secretion.

Milk basic protein: a novel protective function of milk against osteoporosis.

Milk is recommended as an excellent calcium source for bone health. Moreover, milk is considered to contain other components effective for bone health. In our previous studies, using an unfractionated bone cell culture system, we found that milk whey protein, especially its basic fraction (milk basic protein [MBP]), suppressed bone resorption. In this present study, we investigated whether MBP could prevent bone loss in aged ovariectomized rats. Twenty-one 51-week-old female Sprague-Dawley rats were ovariectomized (ovx), and another seven rats received a sham operation (sham). After a 4-week recovery period, the ovx rats were separated into three groups, and they were then fed a control diet, a 0.01% MBP diet (0. 01% casein of the control diet replaced with MBP), or a 0.1% MBP diet for 17 weeks. The sham rats were fed the control diet. Bone mineral density (BMD) of the femur was measured by dual-energy X-ray absorptiometry in vivo. The BMD in the ovx-control group noticeably decreased during the experimental period in comparison with that in the sham group. However, the BMD in the OVX-0.1% MBP group was significantly higher than that in ovx-control group at weeks 12 and 16 (p < 0.05). After the 17-week feeding period, the breaking energy of the excised femur of all groups was determined by use of a three-point bending rheolometer. The breaking energy in the ovx-control group was significantly lower than that in the sham group (p < 0.05). However, the breaking energy in the ovx-0.1% MBP group was significantly higher than that of the ovx-control group (p < 0.05). Urinary deoxypyridinoline (D-Pyr) level of the ovx-control group was higher than that of the sham group, whereas the level of D-Pyr excretion in the ovx-0.01% MBP and ovx-0.1% MBP groups was significantly lower than that of the ovx-control group (p < 0.05). These results suggest that MBP suppresses the osteoclast-mediated bone resorption and prevents bone loss caused by ovariectomy. Moreover, we performed an in vitro study using isolated osteoclasts from rabbit bone to investigate the possible mechanism. MBP dose-dependently suppressed the number of pits formed by these osteoclasts. This result indicates that MBP suppresses bone resorption by its direct effects on osteoclasts. To our knowledge, this study provides the first evidence that MBP directly suppresses osteoclast-mediated bone resorption, resulting in the prevention of the bone loss that occurs in ovx rats.

The effect of verapamil on renal function after warm and cold ischemia in the isolated perfused rat kidney.

The potential usefulness of verapamil, a calcium channel blocker to renal allograft preservation, was investigated using the isolated perfused rat kidney. Two models of ischemic injury were used. In the first model, rat kidneys were exposed to 40 min of 37 degrees C ischemia on the perfusion circuit. Addition of verapamil in doses of 2.5, 5, and 100 microM concentration to the perfusate significantly improved inulin clearance (Cin) and total sodium absorption (TNa) in the hour of reperfusion following ischemia. Regeneration of adenosine triphosphate (ATP) and total adenine nucleotide TAN levels during reperfusion following warm ischemia were also significantly higher in verapamil-treated kidneys. In the second model, rat kidneys were flushed in situ with Collins C2 solution and stored at 0 degrees C for 8 hr. After this period of cold ischemia, they were perfused on a perfusion circuit with perfusion media. Verapamil 2.5 microM was absent from both flush and perfusate (control), or added to just the flush, both the flush and perfusate, or just the perfusate. Addition of verapamil to the flush or the flush and perfusate significantly improved Cin, urine flow rate (V) and TNa during reperfusion, compared with control. Addition of verapamil to just the perfusate did not effect Cin, TNa, or V but did significantly increase RPF. These findings suggest the verapamil may protect against organ damage occurring during both warm and cold ischemia in the absence of any systemic effects and thus may be useful for renal allograft preservation.

Effect of troglitazone (CS-045) and bezafibrate on glucose tolerance, liver glycogen synthase activity, and beta-oxidation in fructose-fed rats.

To clarify the relationship between lipid and glucose metabolism abnormalities in fructose-fed rats, we examined whether an improvement of insulin sensitivity by troglitazone (CS-045) or a decrease in plasma lipids by bezafibrate affects the relationship between serum levels of lipid and glucose. In addition, we also examined changes in liver glycogen metabolism and beta-oxidation in fructose-fed rats. Troglitazone ameliorated fasting hyperlipidemia, hyperglycemia, and hyperinsulinemia. In addition, it augmented glycogen synthase activity by 53%, and decreased the mitochondrial palmitic acid beta-oxidation rate and ketone body production rate by 27% and 55%, respectively. However, hyperglycemia and liver glycogen synthase activity were not improved by bezafibrate treatment despite a marked reduction of serum triglyceride (TG) levels resulting from a 1.76-fold increase in mitochondrial oxidation and a 2.04-fold increase in hepatic ketone body production. These results suggest that abnormalities in glucose and lipid metabolism in fructose-fed rats, which are ameliorated by troglitazone, may be closely linked to reduced glycogen synthase activity in the liver.

Effect of dietary calcium on renal prostaglandins.

The present study was designed to clarify the possible role of renal prostaglandins (PGs) on blood pressure (BP) regulation during calcium (Ca) restriction or supplementation. Twelve normotensive women with a mean age of 21.2 years participated in the study. After 1 week of normal Ca intake (mean +/- SE, 536 +/- 2 mg/day), a low-Ca diet (163 +/- 1 mg/day) was given for a further 1 week. Additional asparagine Ca (3 g as Ca/day) was also given to half of the subjects. BP, heart rate, and serum total and ionized Ca concentrations were measured at the end of each period. Levels of Ca, sodium, PGE2, 6-keto-PGF1 alpha and thromboxane (TX) B2 excreted into urine were also determined. The plasma level of ionized Ca was significantly increased without any change in total Ca in both groups. Low and high Ca intake decreased and increased urinary Ca excretion by 28% and 56%, respectively. BP was not altered after Ca deprivation or loading. However, urinary PGE2 excretion was significantly augmented from 668.9 +/- 68.1 to 959.7 +/- 183.1 ng/day by Ca loading, whereas Ca deprivation decreased PGE2 excretion (695.4 +/- 108.1 to 513.2 +/- 55.2 ng/day). No changes were observed in 6-keto-PGF1 alpha or TXB2 urinary excretion. These results suggest that renal PGE2 synthesis is stimulated or decreased by 1-week Ca loading or deprivation, indicating a possible antihypertensive role of renal PGE2 during high-Ca intake in hypertensives.

Effect of alpha 1-blockade on diminished forearm blood flow in diabetics.

An increased risk of atherosclerotic disease has been reported in patients with diabetes mellitus. The present study was therefore designed to determine forearm blood flow (FBF) in patients with essential hypertension or those with diabetes mellitus with or without hypertension. FBF determined by venous occlusion plethysmography decreased with age in controls as well as in patients with essential hypertension, whereas FBF in diabetics was significantly lower irrespective of age or blood pressure. As a result, vascular resistance was significantly higher in diabetics than in controls or patients with essential hypertension. Glycemic control in normotensive diabetics during 3 weeks significantly augmented a diminished FBF. alpha 1-Blockade by oral administration of 1 mg of prazosin also augmented the diminished FBF in diabetics, in association with a significant decrease in mean blood pressure and vascular resistance. These results suggest that FBF may be a simple and useful index for determining arterial and/or venous distensibility, and that alpha 1-blocker therapy, in addition to glycemic control, may be a first-line antihypertensive treatment for diabetics with associated hypertension.

Hypercalcemia accompanied by hypothalamic hypopituitarism, central diabetes inspidus and hyperthyroidism.

We present here a case of prominent hypercalcemia accompanied by hypothalamic tumor and Graves' disease. A 24-year-old man with hypothalamic tumor showed hypopituitarism, central diabetes inspidus (DI) and hyperthyroidism. Nausea, loss of thirst and appetite, and general fatigue were found with the unveiling of hypercalcemia and hypernatremia. Parathyroid hormone (PTH) and 1alpha-dihydroxyvitamin D levels were suppressed with a normal range of PTH-related protein values. One-desamino-(8-D-arginine)-vasopressin (DDAVP) and half-saline administration normalized hypernatremia, while hypercalcemia was still sustained. Administration of cortisone acetate and thiamazole reduced the elevated serum Ca level. In the present case, concurrent hyperthyroidism was assumed to accelerate skeletal mobilization of calcium into the circulation. Hypocortisolism and central DI was also considered to contribute, to some extent, to the hypercalcemia through renal handling of Ca.

[Clinical features of Japanese children infected with Cryptosporidium parvum during a massive outbreak caused by contaminated water supply].

A massive outbreak of cryptosporidiosis occurred at a local town of Saitama Prefecture, in 1996. During this outbreak, we investigated the clinical features of children seen at Saitama Medical School. Cryptosporidium parvum (C. parvum) was detected from 10 out of 28 (36%) children with diarrhea during June and August, 1996. The average ages of the children who were positive and negative for C. parvum were 6.5 and 5 year old, respectively. Among the children infected with C. parvum, colic pain was observed in 3 children and 4 children had vomiting. However, none of the children showed fever over 38.0 degrees C nor bloody stools. Family members of children infected with C. parvum also had diarrhea and/or vomiting (5/6). C. parvum was repeatedly detected from 2 out of 3 children. All infected children had an improvement of abdominal symptoms in 4 to 10 days. C. parvum should be included as a pathogen which causes enterocolitis in Japanese children.

[Calcitonin].

Calcitonin is a potent inhibitor of osteoclastic bone resorption and has been widely used for the treatment of osteoporosis. Nasal calcitonin, instead of injectable form, is more popular in Europe and United States, while only injectable form has been approved in Japan. The regimen, dose, frequency is remarkably different from study to study, and the standard regimen has not been established for osteoporosis. Fifty to 100 units of salmon calcitonin has been used daily intramuscularly in Europe. Recent trial using nasal calcitonin has shown the similar effects on the bone as the injectable form although the actual resorptionis not so high. In Japan, once weekly 20 units if eel calcitonin analogue injection has been approved for osteoporosis. After administration in the form of either nasal or injectable preparation, peak serum concentration reaches more than 100 pg/ml, far exceeding 10(-11) M, at which level osteoclast bone resorption is rapidly impaired with disappearance of actin ring formation. It is reflected by the decrease of urinary pyridinoline cross-links excretion. Consecutive treatment with calcitonin reduces the calcitonin receptors on the surface of osteoclasts as well as osteoclast precursors, while they are still TRAP positive, suggesting that they retain bone resorbing activity. That may be one of the mechanisms of escape phenomenon. We are not sure whether daily administration of calcitonin can avoid the escape phenomenon and can maintain the bone volume. The standard preparation should be determined by the longer clinical trials with new bone markers and bone mass measurement as the endpoints.

The interaction of calcitonin gene-related peptide with angiotensin II on blood pressure and renin release.

Calcitonin gene-related peptide (CGRP) has been shown to be a potent vasorelaxant. We tested the interaction of CGRP with angiotensin II (Ang II) in conscious, unrestrained Wistar rats. Rat CGRP (rCGRP, 0.1 and 1.0 nmol/kg per min) dose-dependently lowered mean arterial blood pressure and increased the heart rate. The effects continued throughout the infusion period of 30 min. Moreover, rCGRP significantly attenuated the pressor responses to Ang II (100 ng/kg per min). High-dose rCGRP (1.0 nmol/kg per min) almost abolished the pressor action of Ang II, and a much higher dose of Ang II (1000 ng/kg per min) was needed to restore the pre-infusion pressure. Plasma renin activity was dose-dependently increased by rCGRP, but was attenuated by simultaneous Ang II infusion. Rat CGRP also increased the plasma aldosterone concentration, as did Ang II. These results suggest that CGRP may have a neuromodulatory role in cardiovascular regulation.