Dihydrotachysterol Research Articles

Serine phosphate, threonine phosphate and γ-carboxyglutamic acid in normal and experimentally induced, pathologically calcified rat skin (topical cutaneous calciphylaxis)

The amount of non-collagenous proteins is increased greatly during the pathological calcification of rat skin experimentally induced by dihydrotachysterol (DHT) and Ovalbumin (topical cutaneous calciphylaxis). This is accompanied by an increase in the total amount and concentrations of protein-bound serine phosphate [Ser(P)], threonine phosphate [Thr(P)] and gamma-carboxyglutamic acid (Gla), almost all of which can be extracted from the tissue and can be dissociated from collagen in 0.5M EDTA. The EDTA-soluble, non-collagenous proteins are rich in aspartic and glutamic acids, similar to the non-collagenous, EDTA-soluble proteins of bone, cementum and calcified cartilage, and quite distinct from those of dentin and enamel.

The histological changes of alveolar bone, cementum and periodontal fiber due to experimental progeria-like syndrome of rat (author's transl)

Disturbance due to the long term administration of Dihydrotachysterol (DHT) are known on the experimental to cause a progeria-like syndrome.In the present study, DHT was administered once daily for 28th days with 50μg per 100g weight of Sprague-Dawley female rats weighing 180-220g by the tuberculin syringe and attached stomach tube, and traumatic occlusion was experimentally induced by loading abnormal forces to rats.The rats were divided into 4 groups; 1) control group, 2) DHT administered group, 3) DHT administration plus traumatic occlusion group, 4) Traumatic occlusion group.The traumatic occlusion was induced by the method of Hieda and Ikeda.In Group 2 and 3, the animal weight showed a trend of decrease until the 5th day of experiment but after the both groups were maintained with same weight and Group 1 and 4 showed almost linear increase until the 28th day of experiment.The rats administered with DHT showed a tendency of almost atrophy of skin, kyphosis and atrophy of skelton.According to the DHT administration, serum calcium level was markedly increased and serum phospholic acid or mandibular bone calcium were reduced.On histological changes in Group 2 or Group 3, the eosinophilic substance like a layer or hematorylino-philic substance on the surface of alveolar bone or comentum.This substance was recognized like inmineralized tissue (Cementoid, Osteoid) and was markedly seen at the root apex area or root central area and and especially, a periodontal space showed a tendency of narrowing at the above area and arrangement of the periodontal fiber was irregular.In Group 3, changes of the periodontal structure showed marked histological changes in the central area of buccal root surface, a periodontal space was narrowed on the compressed side and widend on the traction side and alveolar bone of the compressed side showed osteoclastic resorption and almost were not observed eosinophilic substance, but in periodontal space of the traction side was observed a little osteoid formation and in generally eosinophilic substance formation at root apex area or bifurcation area were reduced compaired with Group 2.

Calcitriol dosage in osteomalacia, hypoparathyroidism and attempted treatment of myositis ossificans progressiva.

Mineral retention was measured during 39 metabolic balance studies in 34 patients with nutritional osteomalacia or late rickets; they were divided into 5 treatment groups consisting of oral vitamin D, artificial ultra-violet irradiation, 25-hydroxycholecalciferol (calcifediol), 1 alpha-hydroxycholecalciferol (alfacalcidol) and 1 alpha, 25-hydroxycholecalciferol (calcitriol). With the 1 alpha-hydroxylated derivatives, initial dosage of 2 to 6 micrograms daily was required to achieve optimal healing rates by comparison with other responses. Mineral retention was markedly enhanced by supplementation with microcrystalline hydroxyapatite compound (MCHC); untreated X-linked hypophosphataemic rickets healed in 7 weeks on 10 micrograms alfacalcidol daily and 6 grams MCHC daily without developing hypercalcaemia. By contrast, adult-presenting hypophosphataemic osteomalacia developed early hypercalcaemia on the same treatment; additional phosphate supplementation, without changing other treatment, abolished hypercalcaemia and improved calcium retention. A long-term crossover trial of the vitamins D in 6 patients with hypoparathyroidism suggested that relative potencies were as follows (assigning to vitamin D an arbitrary potency of l): vitamin D2 (or D3) l: dihydrotachysterol (DHT) 3: calcifediol 10: alfacalcidol 750: calcitriol 1500. The two-fold superiority of calcitriol over alfacalcidol was evident. Calcifediol and vitamin D controlled plasma calcium at comparable levels of circulating 25-hydroxyvitamin D (25-OH-D), elevated 25-OH-D persisting at least 1 to 2 years after discontinuing long-term (greater than 4 years) vitamin D. In 2 patients with myositis ossificans progressiva treated with 10 to 20 micrograms calcitriol daily, hypercalcaemia was minimized by a low-calcium diet supplemented with cellulose phosphate, suggesting that bone resorption did not play a major role in vitamin D intoxication. Net mineral loss was documented in a young male patient but not in a menopausal female, suggesting that calcitriol treatment was not likely to produce post-menopausal osteoporosis.

Calcium metabolism in bone disease: effects of treatment with microcrystalline calcium hydroxyapatite compound and dihydrotachysterol.

Microcrystalline calcium hydroxyapatite compound (MCHC) was given orally together with small doses of dihydrotachysterol (DHT) to a number of patients with osteogenesis imperfecta (OI). Serial calcium and phosphate balances in three patients representing wide variations in severity of OI are presented over periods from eight months to two years. The combination of MCHC and DHT resulted in an immediate positive calcium balance which was maintained throughout the period of assessment in 2 cases. However, no radiological improvement could be demonstrated. Substituting calcium gluconate for MCHC resulted in a reduction of positive balance. No adverse effects were noted. The reasons why MCHC with DHT should result in increased calcium retention are discussed. This combination of MCHC and DHT could be of benefit in many common situations of bone demineralization, such as osteoporosis.

Examination of dihydrotachysterol-induced progeria as a model for aging changes in carbohydrate metabolism

Interest in the pharmacological effects of drugs in the elderly has created a need for a laboratory model in which responses of aged animals to drugs can be studied. Dihydrotachysterol (DHT)-induced progeria, a syndrome with symptoms similar to those seen in normal aging, was evaluated as an old age model. DHT-treatment was shown to produce a decreased carbohydrate tolerance that was due to an impaired release of insulin from pancreatic islets and not due to a decreased sensitivity to insulin. These changes were unlike those observed with aging. Thus, DHT-induced progeria would not appear to be a good model for aging for the investigation of carbohydrate metabolism. Evidence is presented which indicates that glucose and tolbutamide act via different mechanisms to stimulate insulin release.

The response to 1,25-dihydroxycholecalciferol and to dihydrotachysterol in adult-onset hypophosphataemic osteomalacia

The biochemical changes observed in a patient with adult-onset hypophosphataemic osteomalacia after three weeks treatment with 1,25-dihydroxycholecalciferol (1,25-(OH) 2D 3) followed by dihydrotachysterol (DHT) are reported. The treatment with 1,25-(OH) 2D 3 resulted in restoration of intestinal phosphate absorption to normal with a small rise in plasma phosphate concentration; there was no significant change in tubular reabsorption of phosphate. The tubular reabsorption of bicarbonate, which was initially low, returned almost into the normal range with normalisation of plasma bicarbonate concentration. Aminoaciduria decreased. There were no changes in plasma or urinary calcium but immunoreactive parathyroid hormone (i-PTH) which was initially elevated fell but still remained above the normal range. These changes were maintained after replacing the 1,25-(OH) 2D 3 treatment with dihydrotachysterol (DHT).

Electron microscope tracing of acid mucopolysaccharides after administration of dihydrotachysterol

Nonspecific calcinosis engendered by dihydrotachysterol ( DHT ) is associated with massive extraosseous calcium deposits, in connection with which acid mucopolysaccharides ( AM ) can be observed. Cardiac muscle and the vascular system are particularly affected. The acri-flavine-phosphotungsten acid ( PTA ) contrast-method of tracing AM is based on the affinity of acriflavine for basophile material in the tissue. An electron dense contrast is achieved by means of the complex of acriflavine, which is selectively bound in the tissue, and PTA.Six month old albino rats were sacrificed 1, 2, 3, 5, 8, 10, 13, 17 and 20 days after per os-administration of 3. 0 mg DHT. Tissue samples from cardiac muscle and aorta were first fixed in 2 % glutaraldehyde phosphate-buffer, pH 7. 0 for two hours and then pre-incubated in darkness with 2. 0 x 10-2 M acriflavine in 50 % ethanol phosphate-buffer overnight.

Effect of oral administration of calcium carbonate, aluminum hydroxide gel and dihydrotachysterol on renal acidosis.

The effects of oral administration of calcium carbonate, aluminum hydroxide gel, dihydrotachysterol (DHT) and sodium bicarbonate on metabolic acidosis and plasma calcium and phosphate were studied in 7 patients with chronic renal failure. Single administration of calcium carbonate alleviated the acidosis and increased the urinary bicarbonate excretion. These effects were potentiated when aluminum hydroxide gel was administered in combination with calcium carbonate. The plasma calcium was increased by this combination therapy. The effects of these two agents on acidosis and plasma calcium were further enhanced by the additional administration of DHT. Urinary bicarbonate excretion was less during the treatment with aluminum hydroxide gel and calcium carbonate than with aluminum hydroxide gel and sodium bicarbonate, when the excretions were compared at the similar concentrations of plasma bicarbonate. Aluminum hydroxide gel and DHT are likely to enhance the effect of calcium carbonate, which works as an alkalinizing salt on acidosis, probably through increasing calcium absorption in the intestine. And the three agents suppress the leak of bicarbonate into the urine contributing to the improvement of acidosis.

VITAMIN D RESISTANT HYPOPARATHYROIDISM - IMPAIRED RESPONSE TO 1,25 DIHYDROXYCHOLECALCIFEROL (1,25 DHCC)

Resistance to vitamin D is an unusual, frustrating complication of hypoparathyroidism. Magnesium depletion, coincident malabsorption, ↓hepatic or renal hydroxylation, and ↓Ca++ mobilization from bone are postulated mechanisms, but do not explain relative resistance to all vitamin D sterols, including 1,25 DHCC in three patients recently encountered at two institutions.A.G., a 14 year old calciferol-resistant boy with idiopathic hypoparathyroidism, candidiasis and mild malabsorption, maintained for 4 years with oral Dihydrotachysterol (DHT), 4-6 mg/day, suddenly became refractory to DHT in doses up to 15 mg, daily. Oral 1,25 DHCC to 10 μg was ineffective once daily and unaltered by parenteral Mg SO4. A calcemic response to low dose (20-100 U.) PTH was observed. Response to 1,25 DHCC was achieved only with frequent administration (3 μg. q. 6 hours) + 30 Cm elemental Ca++/day. Two other hypoparathyroid patients, one surgical, have been found resistant to calciferol, DHT and ordinary doses of 1,25 DHCC. One responded to 5 μg once daily and has stabilized with 1-2 μg/day, while the other responds to a 5 μg daily dose only with IM parathormone, 20-40 U./day.Plasma 1,25 DHCC (Dr. H. DeLuca) was normal in all 3 patients immediately following ingestion. Preliminary data in A.G. showing a rapid decline in plasma 1,25 DHCC suggest that accelerated sterol metabolism may account for his apparent “resistance”.

Separation and Ultraviolet Absorbance Techniques for Determining Dihydrotachysterol in Pharmaceutical Preparations

Three separation techniques and ultraviolet (UV) spectrophotometry are described for dihydrotachysterol (DHT). The absorbance procedure eliminates the necessity of establishing a regular baseline, which is advantageous in certain cases. The relative absorbance values obey Beer's law in the range of 1-10 mug DHT/ml ethanol. An absorbance ratio check ensures the applicability of the method with respect to the degree of allowable interference present. The absorbance ratio values at the indicated wave-lengths at this concentration range and in the absence of any interferences are relativiely constant. The UV absorbance could serve as the common final determinative step for the other techniques, either singly or in combination, for quantitating DHT in preparations that require varying degrees of cleanup through extraction, saponification, and thin layer chromatography. The respective mean per cent recoveries and their relative standard deviations for a 50 mug DHT standard sample treated by the 3 techniques are 99.6 -+/ 0.5, 98.5 -+/ 1.1, and 97.3 -+/ 3.3%. Content uniformity determination of DHT in tablets and in soft gelatin capsules is also presented, and precautions are discussed.

Infantile rickets with server proximal renal tubular acidosis, responsive to vitamin D.

A variant of the Fanconi syndrome is reported in an infant with rickets, hypophosphataemia, severe `proximal9 renal tubular acidosis, and generalized hyperaminoaciduria. Noteworthy was the absence of glycosuria and cystinosis. Large doses of dihydrotachysterol (DHT) and of alkali controlled all of these abnormalities. Renal function tests including glomerular filtration rate, renal blood flow, ammonia secretion, titratable acidity, and bicarbonate handling were normal when the child was receiving DHT. At age 4½ years total relapse followed the cessation of therapy, and at this time serum parathyroid hormone levels were not raised. Now DHT alone (without alkali supplement) reversed all of the biochemical abnormalities including the acidosis. This child thus has an apparently vitamin D-dependent `proximal9 tubular acidosis, which is not due to secondary hyperparathyroidism.

The therapeutic approach to hypoparathyroidism

Hypoparathyroid patients have for decades been subjected to vitamin D therapy, in varying pharmacologic doses, with widely discrepant responses. They have been hypocalcemic on extremely large doses of the vitamin and hypercalcemic on doses that are not toxic to normal persons. The resistance to conventional forms of vitamin D and calcium supplementation therapy has produced a need for other therapeutic preparations. Dihydrotachysterol (DHT), 25-(OH)D3 and 1α-(OH)D have been tried in short-term studies, often with limited success. More detailed analyses of long-term results of therapy with 25-(OH)D, 1,25-(OH)2D or 1α-(OH)D are warranted before their superiority over vitamin D and DHT therapy can be established. Until such time that these substances are available in sufficient quantities for these studies, a rational approach to vitamin D therapy should include a more definitive analysis of the factors leading to the hypocalcemic hyperphosphatemic state. This knowledge should be combined with an appreciation of the effects of emotion and psychologic stress, estrogens, oral contraceptives, tranquilizers, anticonvulsants, magnesium deficiency, dietary indiscretions and diuretics on calcium homeostasis. Measurements of circulating PTH levels and para-thyroid reserve, and reservations about accepting urinary values as estimates of circulating calcium levels in treated patients would add additional sophistication to planned therapeutic approaches with vitamin D. The final result of these combined endeavors may ultimately lead to better control and a much happier patient.

The organic-inorganic relationship in calcified mitochondria.

Experimentally induced calcification within mitochondria has been studied electron rnicroscopically. Cells investigated comprise hepatic cells damaged by CCl(4) intoxication, myocardial cells damaged by prolonged dihydrotachysterol (DHT) administration, and cells from skeletal muscle (gastrocnemius) damaged by DHT sensibilization and local injury. Cells from a human bowel carcinoma were studied too. Two types of intramitochondrial inorganic inclusion have been found. The first consists of clusters of apatite-like, needle-shaped crystals (crystalline aggregates), the second of clusters of very fine granules (granular aggregates). The former have been found mainly in mitochondria in apparently normal myocardial and muscular cells, the latter in mitochondria of degenerated hepatic, neoplastic, and myocardial cells. Crystalline aggregates are closely related to the membranes of cristae at first, but they later spread to occupy the whole mitochondrial matrix. Granular aggregates are initially found in the mitochondrial matrix near, but perhaps not touching, cristae; by growing they come into close contact with cristal membranes. Both types of aggregate show intrinsic electron opacity, which disappears after formic acid decalcification. Only the crystalline aggregates give an electron diffraction pattern of crystallinity. Uranium and lead staining of decalcified sections shows that both types of aggregate are intimately connected with an organic substrate. The substrate of crystalline aggregates consists of very thin, elongated structures shaped like the inorganic crystals. The substrate of granular aggregates consists of amorphous material gathered in clusters, with the same roundish shape and intercristal position as the inorganic granules. Both types of substrate are stained by phosphotungstic acid at low pH and by silver nitrate-methenamine after periodic acid oxidation. These results show that the organic content of the substrates includes glycoproteins; they have been confirmed by the periodic acid-Schiff (PAS) method under the optical microscope. These findings have been discussed in relation to the recent discovery of organic Ca(2+)-binding sites in mitochondria and to the general problems of soft tissue calcification.

Vitamin D-dependent rickets: actions of parathyroid hormone and 25-hydroxycholecalciferol.

Extract: During active rickets, the increase in 3′,5′-adenosine monophosphate (3′,5′-AMP) excretion after infusion of parathyroid hormone (PTH) can be claimed as a demonstration of PTH responsiveness, at least as far as the kidney is concerned (Table II). In patients with vitamin D-dependent rickets, following 25-HCC therapy, the basal excretion ratios of nanomoles 3′,5′-AMP/milligrams creatinine fall within the normal range, and, although the basal ratios of milligrams phosphate/milligrams creatinine excretion decrease to normal (Tables III, IV, and V), a phosphaturic response to PTH is still not evident. The high basal ratios of nanomoles 3′,5′-AMP/ milligrams creatinine and milligrams phosphate/milligrams creatinine suggest underlying hyperparathyroidism during a phase of this syndrome marked by hypocalcemia and hyperaminoaciduria. During 2-day PTH challenges, while rickets was active, the peak increases in serum calcium above base line were 1.92, 1.80, and 1.86 mg/100 ml. After treatment with 25-hydroxycholecalciferol (25-HCG), no further significant increases in serum calcium above base line could be elicited (Tables VI, VII, VIII). It would seem, therefore, that, while these patients were untreated, small but sufficient amounts of 25-HCC or another metabolite of vitamin D were available to permit the calcium-mobilizing action of PTH on bone. The observed hypocalcemia in vitamin D-dependent rickets, then, is most likely secondary to a defect in gastrointestinal absorption. Mineral balance studies during active rickets revealed an absorptive defect for calcium, phosphorus, and magnesium—a defect completely reversible after 25-HCC treatment. Speculation: We suggest that the small intestine represents the prime target organ site for impaired vitamin D action in this disease by differential failure in the biologic formation of 1,25-dihydroxycholecalciferol from 25-HCC. Similarly, we postulate that a variable hydroxylating deficiency may be present in the transformation of vitamin D3 to 25-HCC. Genetic heterogeneity, therefore, may ultimately explain common responses in these three patients to contrasting doses of dihydrotachysterol (DHT) and 25-HCC.

Effect of Dihydrotachysterol on TSH Secretion in Rats

Crystalline dihydrotachysterol (DHT) stimulates the activity of the thyroid gland in puberal female rats. After i.p. injection of 350 or 500 µg/kg/day DHT, the height of epithelial thyroid cells increased, resulting in an increase of thyroid cells from 19 to 26% of the total follicle area. The secretory activity of the gland increased, serum TSH rising from 1.1 to 2.1 mU/ml and serum thyroxine rising from 2.5 to 6 µg%. The basal metabolic rate increased from 1,400 to 1,810 ml O2/kg/h oxygen consumption. Body weight, however, did not differ from that of the control group. At a dosage of 750 µg/kg/day, the above reaction was accompanied by a body weight drop of 20%. Within 4 h after implantation of crystalline DHT into the anterior basal hypothalamus, the thyroid reacted histologically with the above pattern of an active gland. These changes could not be elicited in hypophysectomized rats. These results would indicate that the stimulatory effect of DHT on the thyroid gland acts via the hypothalamo-pituitary axis.

The mineralization of hair follicle tissue. I. An in vivo study.

Previous histological investigations have shown that the hair follicle is particularly susceptible to mineralization when the skin of hypercalcaemic rats is injured. Direct chemical and X-ray diffraction analyses of follicle tissue have now confirmed this finding. As judged by increases in both calcium and phosphorus, mineral deposits began to form in hair follicle tissue 6–12 h after a mild crush injury to the skin of rats dosed with dihydrotachysterol (DHT), and 24–48 h after a similar injury to the skin of non-dosed rats. X-ray diffraction gave a diffuse apatite pattern. Within 3 h of injury there was a rise in the calcium content of follicle tissue which was not related to DHT-dosing and which was probably a reflection of calcium binding rather than mineral deposition.

Effect of heat on serum thyroxin and its modification by dihydrotachysterol.

Dehydrotachysterol (DHT 500 µg/kg/24 h) erhoht den TSH- und T-4-Spiegel in Rattenserum bei normalen und hohen Temperaturen, ohne den Calcium-Spiegel zu verandern, und verhindert den Tod von Ratten, die bei 34° oder 37°C gehalten werden. Das Uberleben der Ratten nach DHT-Injektionen erklart sich durch erhohte T-4-Produktion, die einen negativen «Feedback» auf den Hypothalamus ausubt und so die lethale Uberproduktion von TSH verhindert.

Estrogen inhibition of a progeria-like syndrome in the rat.

Abstract:Forty female Wistar‐Imamichi rats, each weighing 98 gm, were divided into 5 equal groups and the effects of dihydrotachysterol (DHT) on the calcium content and weight of the kidney, heart, aorta and femur were compared in ovariectomized and in intact rats. The effect of DHT was much more pronounced in the ovariectomized than in the intact rats. These findings strongly suggest the protective effect of endogenous estrogens on the DHT‐induced progeria‐like syndrome in rats.

Influence of various catatoxic steroids upon chronic dihydrotachysterol intoxication.

In rats, the catabolism normally induced by chronic dihydrotachysterol (DHT) intoxication is counteracted (in approximately decreasing order of activity) by: norbolethone, ethylestrenol, CS-1 and pregnenolone-16α-carbonitrile (PCN). Spironolactone was previously shown to antagonize DHT in acute experiments. However in the chronic experiments reported here, it is inactive in this respect and even abolishes the high anti-DHT of norbolethone and ethylestrenol. Several possible mechanisms are discussed which might explain these antagonisms, for example: (1) Spironolactone may act by accelerating the enzymatic degradation of the protective anabolic steroids. (2) The 2 types of steroids may compete with each other for receptor sites. (3) Spironolactone and the anabolics may exert opposing actions on DHT metabolism.

Protection by catatoxic steroids against dihydrotachysterol intoxication

Experiments in the rat indicate that both the mortality and the cardiovascular and renal calcification induced by acute intoxication with a single massive dose of dihydrotachysterol (DHT) are inhibited by a variety of steroid hormones and hormone derivatives. In this respect, the most active compounds are those that had previously been shown to exhibit a similar catatoxic activity against various other drugs. No close correlation has been found between other hormonal effects or the chemical structure of the catatoxic steroids and their ability to protect against acute DHT-intoxication. It is noteworthy however, that all steroids that proved to be highly active in raising resistance to DHT were also endowed either with anti-mineralocorticoid or with anabolic properties.