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Identification of 7-dehydrocholesterol, vitamin D3, 25(OH)-vitamin D3 and 1,25(OH)2-vitamin D3 in Solanum glaucophyllum cultures grown in absence of light.

Solanum glaucophyllum contains the calciotropic hormone 1, 25-dihydroxy-vitamin D3 (1,25(OH)2D3). The metabolic pathway leading to the formation of 1,25(OH)2D3 in the plant is largely unknown. Specifically, there is controversy about the participation of a photolytic reaction in the generation of vitamin D3 and its metabolites. To investigate the requirement for light, S. glaucophyllum tissue (callus) and cell suspension cultures grown under strict conditions of darkness were extracted with chloroform/methanol (1:2, v/v) followed by purification of the lipidic fraction by Sephadex LH-20 and high-performance liquid chromatography. HPLC peaks with elution times similar to those of authentic samples of 7-dehydrocholesterol, vitamin D3, 25(OH)D3 and 1,25(OH)2D3 were detected. The presence of 1,25(OH)2D3 was also evidenced by [3H]1,25(OH)2D3 competitive binding analysis using the chick hormone intestinal receptor. Furthermore, 7-dehydrocholesterol, vitamin D3, 25(OH)D3 and 1,25(OH)2D3 were unequivocally identified by mass spectrometry. Incubation of control samples of 7-dehydrocholesterol under the same conditions as S. glaucophyllum cultures did not result in vitamin D3 formation, excluding the influence of light in these experiments. The results suggest that a synthetic route of vitamin D3 compounds independent of light operates in Solanum glaucophyllum cultured in vitro.

Argentina↗

Effect of culture conditions on the synthesis of vitamin D(3) metabolites in Solanum glaucophyllum grown in vitro.

In cultured Solanum glaucophyllum we have recently described the operation of a light-independent pathway of 1alpha,25-dihydroxy-vitamin D(3) (1alpha,25(OH)(2)D(3)) biosynthesis which involves similar intermediates as in vertebrates. In this work we investigated factors influencing the formation of 1alpha,25(OH)(2)D(3) and related sterols in S. glaucophyllum grown in vitro in darkness. Callus tissue and cells cultured in Murashige and Skoog medium in the absence of light were employed. Lipids were extracted with chloroform-methanol. The remaining water soluble fraction was incubated with beta-glucosidase to release vitamin D(3) compounds from their glycoconjugated derivatives followed by organic solvent extraction. Vitamin D(3) derivatives were isolated by Sephadex LH-20 and high-performance liquid chromatography (HPLC). HPLC or competitive protein binding assays with intestine 1alpha,25(OH)(2)D(3) receptor and serum vitamin D binding protein were used to quantify the metabolites. The levels of 1alpha,25(OH)(2)D(3) in calli varied according to the tissue explant origin, e.g. stem>leaf>fruit. For all organs, the metabolite was mainly present as free sterol (>80% of total). There were larger amounts of 25(OH)D(3) than 1alpha,25(OH)(2)D(3). It was found that Ca(2+), auxin and kinetin are important factors upregulating 1alpha,25(OH)(2)D(3) synthesis in S. glaucophyllum tissue and cells. Irradiation with UV light increased vitamin D(3) but not 1alpha,25(OH)(2)D(3) levels. In agreement with these results, incubation of cells with [3H]25(OH)D(3) revealed a low conversion rate to [3H]1alpha,25(OH)(2)D(3). The operation of a light-dependent pathway formation of vitamin D(3) coupled to higher expression of 25(OH)D(3)-1alpha-hydroxylase may account for the large concentrations of 1alpha,25(OH)(2)D(3) normally found in differentiated field-grown plants.

Calcifediol↗

1,25(OH)2 vitamin D concentration in the plasma of Solanum glaucophyllum intoxicated rabbits.

OBJECTIVE: To test Solanum glaucophyllum calcinotic effects in adult New Zealand White rabbits in relation to cumulative dose and active principle concentration in plasma. DESIGN: An intoxication assay with controls. PROCEDURE: Rabbits were orally dosed with aqueous extracts of dry leaves of S glaucophyllum for 5, 7 or 9 days. During the experiment, body weight, calcaemia and phosphataemia were measured; retinal blood vessel calibre was observed by ophthalmoscopic examination of the ocular fundus. 1,25(OH)2 vitamin D plasma concentration was determined at the end of the experimental periods. Soft tissue calcium concentration and the presence of calcinotic lesions were studied after euthanasia. RESULTS: Toxic effects were evident in S glaucophyllum treated groups (loss of body weight, elevation of soft tissue calcium concentration, and presence of calcinotic lesions). Plasma 1,25(OH)2 vitamin D concentrations were negatively correlated with final body weight (r = -0.97; P < or = 0.001), and positively correlated with renal calcium concentration (r = 0.74; P = 0.02). There was also a significant regression of plasma 1,25 (OH)2 vitamin D concentration on the cumulative dose of S glaucophyllum (R2 = 0.87; P < or = 0.001). CONCLUSIONS: The procedure described here offers a sensitive and practical experimental model for the study of the pathogenesis of enteque seco.

Animals↗

Screening of Vitamin D activity (VDA) of Solanum glaucophyllum leaves measured by radioimmunoassay (RIA).

The ingestion of Solanum glaucophyllum (SG) causes a calcinosis of cattle named Enteque Seco (ES). The toxic principle is the 1,25-(OH)(2)D(3), mainly conjugated as glycoside. This study aims to validate a simple novel method of evaluation of the VDA of SG leaves. Aqueous extracts of SG were purified using C(18) minicolumns and assayed by RIA with an antibody raised in rabbits by injection of the acid-C22, 1alpha-(OH)Vitamin D(3). Data were expressed as glycoside equivalent to 1,25-(OH)(2)D(3) in ng/g of dry leaves. We compared this data with 1,25-(OH)(2)D(3) levels measured, in the same samples, by liquid chromatography (HPLC) after enzyme cleavage. This procedure involved the incubation of SG leaves with rumen fluid, followed by C(18)-OH solid phase extraction. The 1,25-(OH)(2)D(3) fraction was run by HPLC and detection was achieved using a photodiode array detector. Data were expressed as micrograms of 1,25-(OH)(2)D(3)/g dry leaves. A significant regression of 1,25-(OH)(2)D(3) levels (Y) as a function of glycoside RIA 1,25-(OH)(2)D(3) equivalents (X) was found: Y=12.02+0.35X [R=0.81; P=0,0002; N=15], allowing us to conclude that this novel assay could be used to estimate the amount of this active principle contained in SG leaves.

Chromatography, High Pressure Liquid↗

Utilizing Solanum glaucophyllum alone or with phytase to improve phosphorus utilization in broilers.

Experiments were conducted to determine if Solanum glaucophyllum (SG), a plant containing a glycoside of 1,25-dihydroxyvitamin D, could be used as a feed additive to improve P utilization of broilers. SG leaves (1, 2.5, or 5 g/kg), 1,25-dihydroxyvitamin D (15 microg/kg), or Ca and P (to achieve a 0.92% Ca:0.65% P:0.41% nonphytate P control diet) were added to a 0.56% Ca/0.45% P/0.28% nonphytate P basal diet and fed to broilers from 7 to 28 d of age. Birds fed basal ration alone exhibited reduced weight gain, bone density, and bone mineral content when compared with birds fed the 0.92% Ca:0.65% P diet. Adding 5 g SG leaves or 15 microg of 1,25-dihydroxyvitamin D/kg to the basal diet increased body weight gain, plasma Ca and P, bone ash, and bone density above basal diet levels. Plasma P and weight gain of birds fed 5 g SG or 15 microg of 1,25-dihydroxyvitamin D/kg basal diet were equivalent to those observed in birds fed the 0.92% Ca:0.65% P diet. In experiment 2, the effect of higher doses of SG, as well as the additive effect of SG with 1,200 phytase units/kg diet, were examined in chicks fed a 0.59% Ca and 0.42% P basal diet. Two levels of SG leaves (7.5 g and 10 g), phytase, or both SG (7.5 g) and phytase were added per kilogram of basal diet. Adding SG or phytase to the basal diet increased weight gain, plasma Ca, plasma P, and bone mineral content over that observed in birds fed basal diet alone. Combining SG with phytase provided no significant gains in growth or bone parameters over treatment with phytase alone.

6-Phytase↗

Macrophages and giant cell proliferation associated with bone protein synthesis and calcification in the trachea and bronchi of rabbits intoxicated with Solanum glaucophyllum.

A histopathologic, immunohistochemical, and ultrastructural study of the trachea and the bronchi of 6 rabbits experimentally intoxicated with the calcinogenic plant Solanum glaucophyllum was performed. Histologically, infiltration of the mucosa and the submucosa of the trachea and the bronchi by macrophages, multinucleated giant cells, a few lymphocytes and mast cells, and calcium deposits in the basal lamina of the epithelium and in elastic fibers were observed. Expression of osteocalcin, osteonectin, and osteopontin was detected in the mucosa, lamina propria, and epithelium. Electron microscopic study of the corresponding areas showed numerous macrophages in the process of fusion to form multinucleated giant cells, activated mesenchymal cells, and calcium precipitation in the basal lamina of epithelium and in elastic fibers. It is suggested that the high levels of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in the plant induces macrophage proliferation, multinucleated giant-cell formation, mesenchymal cell activation, bone-protein synthesis, and calcification. In addition, the synthesis of 1,25(OH)2D3 by local macrophages may have contributed to the calcification.

Animals↗

Calcium absorption by intestine. Stimulation in vitamin D-deficient nephrectomized rats by Solanum glaucophyllum.

The administration of an aqueous extract made from leaves of the South American plant Solanum glaucophyllum increased active duodenal calcium absorption 6-fold in sham operated vitamin D-deficient rats and 4.5-fold in comparable animals that had been nephrectomized. These results demonstrate that the active principle from the plant does not require renal metabolism for effects on intestinal calcium absorption, a biological activity previously observed only with sterols such as 1,25-dihydroxyvitamin D3 which have a 1-alpha hydroxyl group or a steric equivalent. The similarity of action between these lipid soluble sterols and the water soluble plant material may be explained by the presence of glycosidal alkaloidal steroids in plants from the genus Solanum.

Alkaline Phosphatase↗

1alpha,25-dihydroxyvitamin D3-like component present in the plant Solanum glaucophyllum.

The ability of an aqueous extract prepared from dried leaves of the South American shrub Solanum glaucophyllum to interact with a chick intestinal receptor system highly specific for the biologically active form of vitamin D, 1alpha, 25-dihydroxyvitamin D3 (1alpha,25-(OH)2D3), was monitored under both in vivo and in vitro conditions. The pretreatment of chicks with the S. glaucophyllum extract reduced the amount of subsequently administered radioactive 1alpha, 25-dihydroxyvitamin D3 localized in the intestinal chromatin fraction by 90% when compared with vitamin D-deficient controls. In addition, the active principle in the aqueous extract was found to compete effectively with 1alpha, 25-(OH)2D3 in a reconstituted intestinal receptor system in vitro. The plant factor(s) was found to be as effective as 1alpha, 25-(OH)2D3 in decreasing the renal 25-hydroxycholecalciferol-1alpha hydroxylase activity levels when administered to vitamin D-deficient chicks. By means of the in vitro intestinal receptor assay, it was possible to estimate that there are approximately 10 to 60 mg of the biologically active material in one kilogram of dries S. glaucophyllum leaves. These data indicate that the plant factor(s) may be a structural analog of 1alpha, 25-(OH)2D3 and suggest the feasibility of its isolation.

Animals↗

The effects of long term feeding of Solanum glaucophyllum to growing rats on Ca, Mg, P and bone metabolism.

The effects of the long term ingestion of Solanum Glaucophyllum leaves (SG) by the rat was investigated in two series of experiments; the animals were fed a normal (for 8 weeks) or a low Ca diet (for 5 weeks). With both diets, the intestinal absorption of Ca was increased and the endogenous fecal Ca excretion was decreased by SG treatment. Added to a normal Ca diet, SG increased the urinary excretion of Ca, Mg and P and reduced the excretion of hydroxyproline and pyrophosphate. At the histological level, SG induced a higher rate of bone tissue synthesis on trabecular and endosteal surfaces. The bone content of hydroxyproline and citrate increased significantly. The total alkaline phosphatase activity of plasma decreased as a function of SG intake due to a decreased in the activity of the intestinal isoenzyme, which was not compensated by the increase in the bone isoenzyme activity. The Mg absorption was decreased by SG inducing lower Mg balances and lower plasma Mg levels. Added to a low Ca diet, SG increased the severity of the secondary hyperparathyroidism induced by the diet. The urinary excretion of hydroxyproline and the plasma alkaline phosphatase activity (both isoenzymes) were significantly increased. The Na and K content of bone decreased as a function of SG intake. 45Ca kinetic experiments revealed that SG increased the rate of Ca resorption and the rate constant of the fast exchangeable Ca pool, in both diets.

Alkaline Phosphatase↗

Solanum glaucophyllum as source of 1,25-dihydroxyvitamin D3.

Vitamin D-deficient rats given an aqueous extract of the South American plant Solanum glaucophyllum accumulate 1,25-dihydroxyvitamin D3 in their blood and intestines at the time they show enhanced intestinal calcium absorption. The identity of the 1,25-dihydroxyvitamin D3 was established by co-chromatography with 1,25-dihydroxy[23,24-3H]vitamin D3 on Sephadex LH-20 columns, microparticulate silica gel columns, a reversed-phase column developed under high pressure, and by a specific 1,25-dihydroxyvitamin D3 binding assay. The chromatographic systems used are fully capable of resolving all of the known metabolites of vitamin D3. Serum of the S. glaucophyllum-treated rats showed 300 pg/ml of 1,25-dihydroxyvitamin D3 and no detectable 1,25-dihydroxyvitamin D2. Similarly, intestine of such rats had 230 pg/g of 1,25-dihydroxyvitamin D3. Control animals which received the vehicle instead of S. glaucophyllum had only 20 pg/ml of 1,25-dihydroxyvitamin D3 in their serum and 4.4 pg/g of 1,25-dihydroxyvitamin D2 in their intestine. These results demonstrate that S. glaucophyllum extracts must be a source of 1,25-dihydroxyvitamin D3; thus a significant basis for the calcinogenic properties of S. glaucophyllum must be the presence of a conjugated form of 1,25-dihydroxyvitamin D3, which is rendered available by digestion.

Animals↗

Collagenous and elastic system fibres in the aorta of cattle poisoned by Solanum glaucophyllum.

The proportions of fibres of the collagenous and elastic systems were measured in the aortas of three normal heifers and in nine heifers given the calcinogenic plant Solanum glaucophyllum for 15, 30 or 60 days. There were decreases in the amount of collagen relative to reticulin, and in the proportion of elastic fibres which were related to the period of dosing. These changes may have an adverse influence on the animals' cardiovascular function.

Analysis of Variance↗

Presence of estrogens and estrogen receptor-like proteins in Solanum glaucophyllum.

The synthesis of mammalian steroid hormones by plants has been reported. However, their physiological role in plants is controversial. The existence of receptor molecules as those of animal cells could provide clues into a possible steroid mechanism of action. Solanum glaucophyllum callus cultures were found to contain not only 17beta-estradiol and estrone but also abundant estrogen binding sites. These sites were specific for 17beta-estradiol ( approximately 550 fmol/mg protein) and could also be competed by the known estrogen receptor (ER) agonist diethylstilbestrol. Antibodies directed against specific sequences of the classical ER alpha isoform, labelled a approximately 67 kDa band which comigrated with the mammalian ER alpha antigen. ER alpha-like proteins were tested positive as estrogen binders in Ligand blot experiments using 17beta-estradiol macromolecular derivatives as ligands. Our results provide first evidences on the existence of estrogen binding proteins structurally related to the mammalian ER alpha subtype in a higher plant system.

Animals↗

Fetal blood calcium response to maternal hypercalcemia induced in the cow by calcium infusion or by solanum glaucophyllum ingestion.

Bovine fetuses chronically catheterized in utero during the last month of gestation were used to study the relationships of maternal-fetal calcium and phosphate levels. Calcium infusion into the pregnant cows induced a rapid and marked increase in maternal plasma calcium but no change in fetal plasma calcium concentrations. Oral administration of Solanum glaucophyllum to the mother for 6 days raised the plasma concentrations of calcium and phosphate in both the mother and the fetus.

Animals↗

On the nature of arterial and lung calcifications induced in cattle by Solanum glaucophyllum.

The chemical and crystalline composition of the mineral deposited in ossified arterial and lung tissues of cows affected by enteque seco (enteque ossificans) has been investigated. The arterial calcification precedes that of lung. The latter was observed only in animals with a calcification of the aorta above 150 mumoles Ca per gram of wet tissue. The mineral deposited in arterial and lung tissues appeared to be a poorly crystallized apatite; following incineration the diffraction patterns of carbonate apatite and magnesium whitlockite, respectively, were found.

Animals↗

Effects of 1alpha,25-dihydroxyvitamin D3 and Solanum glaucophyllum on intestinal calcium and phosphate transport and on plasma Ca, Mg and P levels in the rat.

The responses elicited by a single dose of either S. glaucophyllum (SG) or 1alpha,25-dihydroxyvitamin D3 (1alpha, 25-(OH)2D3) in vitamin D-deficient rats were qualitatively identical. Both compounds stimulated duodenal absorption of calcium and phosphate 3 h after administration, and the effects persisted for 96 h. The SG effects on calcium transport were maximal at 6 h and exceeded the greatest response to 1alpha,25-(OH)2D3 which occurred at 48 h. While the early SG effects on phosphate absorption exceeded those of 1alpha,25-(OH)2D3, the latter compound had a much greater effect at 48 h. Both SG and 1alpha, 25-(OH)2D3 produced marked hyperphosphatemia that was accompanied by hypocalcemia and transient hypermagnesemia at 3-6 h. The phosphatemia increased for 48 h in the 1alpha,25-(OH)2D3 group and fell toward baseline between 48 and 96 h, while this parameter was maximal in the SG group at 3-12 h and returned to baseline values by 96 h. Plasma calcium levels tended to vary inversely with phosphorus concentrations and did not exceed baseline values until 48 h for the SG group and 96 h for the 1alpha,25-(OH)2D3 group. We suggest that the hypocalcemia is due to calcium phosphate precipitation. The quantitative differences in the effects on calcium and phosphorus metabolism may be dose-related. The responses to SG suggest that a gram of dried leaf has approximately the same activity as 1 mug of 1alpha,25-(OH)2D3. It appears that the water-soluble SG factor has the same biological effects on Ca and P metabolism as does 1alpha,25-(OH)2D3.

Animals↗