Vitamin D2-enriched button mushroom (Agaricus bisporus) improves memory in both wild type and APPswe/PS1dE9 transgenic mice.

Vitamin D deficiency is widespread, affecting over 30% of adult Australians, and increasing up to 80% for at-risk groups including the elderly (age>65). The role for Vitamin D in development of the central nervous system is supported by the association between Vitamin D deficiency and incidence of neurological and psychiatric disorders including Alzheimer's disease (AD). A reported positive relationship between Vitamin D status and cognitive performance suggests that restoring Vitamin D status might provide a cognitive benefit to those with Vitamin D deficiency. Mushrooms are a rich source of ergosterol, which can be converted to Vitamin D2 by treatment with UV light, presenting a new and convenient dietary source of Vitamin D2. We hypothesised that Vitamin D2-enriched mushrooms (VDM) could prevent the cognitive and pathological abnormalities associated with dementia. Two month old wild type (B6C3) and AD transgenic (APPSwe/PS1dE9) mice were fed a diet either deficient in Vitamin D2 or a diet which was supplemented with VDM, containing 1±0.2 µg/kg (∼54 IU/kg) vitamin D2, for 7 months. Effects of the dietary intervention on memory were assessed pre- and post-feeding. Brain sections were evaluated for amyloid ß (Aß) plaque loads and inflammation biomarkers using immuno-histochemical methods. Plasma vitamin D metabolites, Aß40, Aß42, calcium, protein and cholesterol were measured using biochemical assays. Compared with mice on the control diet, VDM-fed wild type and AD transgenic mice displayed improved learning and memory, had significantly reduced amyloid plaque load and glial fibrillary acidic protein, and elevated interleukin-10 in the brain. The results suggest that VDM might provide a dietary source of Vitamin D2 and other bioactives for preventing memory-impairment in dementia. This study supports the need for a randomised clinical trial to determine whether or not VDM consumption can benefit cognitive performance in the wider population.

Intoxicación por sobredosificación de vitamina D en un lactante / Poisoning by overdose of vitamin d in an infant

El objetivo de esta nota técnica es la descripción y comentarios de un caso clínico reciente de intoxicación por sobredosificación por vitamina D, y la correcta interpretación clínica y de los parámetros de laboratorio. Caso clínico. Lactante de 6 meses en tratamiento con Biominol® (suplemento vitamínico), ingresó debido a un estado de decaimiento e irritabilidad. Las analíticas iniciales muestran concentraciones de calcio iónico en sangre de 2,11mmol/L (intervalo de referencia (IR): 1,15-1,29mmol/L), y concentración de calcio total plasmático de 5,5mmol/L (IR: 2,25-2,75mmol/L). En nuestro laboratorio, las vitaminas D2 y D3 fueron determinadas por cromatografía líquida de alta resolución (HPLC), y por un método electroquimioluminiscente, que mide la vitamina D total. Los valores de vitamina D2 fueron 419ng/mL y vitamina D total 482ng/mL (IR: 30-100ng/mL). La intoxicación de vitamina D tuvo origen exógeno, debido al incremento de vitamina D2. El diagnóstico definitivo fue hipercalcemia severa secundaria a intoxicación por vitamina D y nefrocalcinosis secundaria a esta con función renal normal con hipercalciuria. Como conclusión, cabe destacar la importancia de la correcta dosificación de los pacientes y la determinación de las diferentes formas de vitamina D para averiguar su origen, realizando una correcta interpretación (AU)

The objective of this technical note describes and comments on a recent clinical case of poisoning by overdose of vitamin D, and the correct interpretation of clinical and laboratory parameters. Vitamin D is a fat-soluble vitamin involved in the absorption of calcium and phosphorus in the intestine. Administration of high doses for prolonged periods can cause hypercalcemia, leading to kidney failure and renal calcifications. Clinical case. The definitive diagnosis of this patient was severe hypercalcemia secondary to exogenous vitamin D poisoning, and nephrocalcinosis secondary to this with normal renal function with hypercalciuria. In conclusion, the correct dosing of patients and determination of different forms of vitamin D to trace its origin and making a correct interpretation is important. Male, 6 months old in treatment with Biominol® (vitamin D supplement), was admitted to the emergency department because of a state of decline and irritability. The initial analytical results showed an ionized calcium concentration in blood of 2.11mmol/L (reference interval (RI): 1.15 - 1.29mmol/L), and plasma total calcium concentration of 5.5mmol/L (RI: 2.25-2.75mmol/L). In our laboratory, Vitamin D2 and D3 were determined by liquid chromatography high resolution (HPLC), and an electrochemiluminescence method. The results showed a vitamin D2 419ng/mL and total vitamin D 482ng/mL (RI: 30-100ng/mL). It was found that the vitamin D overdose was of exogenous origin, due to increased vitamin D2 (AU)

Differential effects of vitamin D receptor activators on aortic calcification and pulse wave velocity in uraemic rats.

BACKGROUND: Vascular calcification is associated with an increase in cardiovascular mortality in stage 5 chronic kidney disease. To determine if vitamin D receptor activators (VDRAs) have differential effects in the pathogenesis of aortic calcification, we assessed the effects of paricalcitol and doxercalciferol in vivo using 5/6 nephrectomized (NX) rats. To quantify the functional consequences of vascular calcification, pulse wave velocity (PWV), an aortic compliance index, was measured. METHODS: NX rats were fed a diet containing 0.9% phosphorous and 0.6% calcium 4 weeks prior to and throughout the study. On Day 0, rats received vehicle or VDRA (0.083, 0.167 and 0.333 microg/kg, i.p.) three times per week for 6 weeks. At Day 0 and Weeks 2 and 6, blood was drawn and PWV was measured by Doppler ultrasound. RESULTS: VDRAs (0.167 and 0.333 microg/kg) consistently lowered PTH at Weeks 2 and 6. All doses of paricalcitol increased serum calcium at Week 6 but not at Week 2, while the two higher doses of doxercalciferol increased serum calcium at both Weeks 2 and 6. Treatment with paricalcitol (0.333 microg/kg) increased serum phosphorus at Weeks 2 and 6; these changes were not different from those observed in 5/6 NX rats. All doses of doxercalciferol increased serum phosphorus at Week 6. Paricalcitol had no effect on Ca x P; however, the two highest doses of doxercalciferol increased Ca x P at Weeks 2 and 6 above that observed in the 5/6 NX vehicle-treated group. There were no differences in aortic calcium and phosphorus contents at the end of 6 weeks among SHAM-, 5/6 NX- and paricalcitol-treated rats. However, treatment with the two higher doses of doxercalciferol caused a significant elevation in aortic calcium and phosphorus contents. Measurements of PWV demonstrated differential effects of VDRAs on vascular compliance. Paricalcitol produced no effects on PWV, while the two highest doses of doxercalciferol increased PWV at Week 6. CONCLUSIONS: In uraemic rats with established secondary hyperparathyroidism, we demonstrate differential effects of paricalcitol and doxercalciferol on aortic calcification and PWV, independent of serum Ca, P and Ca x P, suggesting different mechanisms of action between VDRAs.

Phase II study of doxercalciferol for the treatment of myelodysplastic syndrome.

We conducted a phase II trial of doxercalciferol, a vitamin D2 analogue, in 15 patients with MDS. Each received doxercalciferol 12.5 microg orally daily for 12 weeks. Nine of 15 patients completed the prescribed course and of these, six had stable disease. No patient had a response (IWG criteria) and overall eight patients experienced progressive disease while on therapy. Two patients with chronic myelomonocytic leukemia (CMML) had a marked rise in monocytes on study. Overall the treatment was well tolerated. One patient was removed from study due to hypercalcemia. We conclude that short-term treatment with doxercalciferol has limited activity in patients with MDS.

Effectiveness of 1alpha-hydroxyvitamin D2 in inhibiting tumor growth in a murine transgenic pigmented ocular tumor model.

OBJECTIVE: To study the effectiveness of the vitamin D analogue 1alpha-hydroxyvitamin D(2) (1alpha-OH-D(2)) in inhibiting ocular tumor growth in transgenic "Tyr-Tag" mice that developed pigmented ocular tumors produced with the simian virus 40 T and t antigens under the control of the mouse tyrosinase gene. These animals develop pigmented intraocular tumors primarily from the retinal pigment epithelium that closely resemble the histologic features and growth pattern of human choroidal melanoma. METHODS: A total of 73 Tyr-Tag transgenic mice between 6 and 7 weeks old were randomly assigned by sex and litter to 3 treatment groups to receive 0.05 microg/d, 0.1 microg/d, or 0.2 microg/d of 1alpha-OH-D(2); a control group received vehicle (coconut oil). The drug was administered by oral gavage 5 times a week for 5 weeks. The animals were then euthanized and their eyes were enucleated and processed histologically. Three serial sections from each eye were examined microscopically and the mean tumor area measured using Optimus software version 6.5 (Media Cybernetics LP, Silver Spring, Md). Toxic adverse effects were assessed on the basis of mortality, weight loss, and serum calcium levels. RESULTS: The mean tumor size in the 0.1- microg/d and 0.2- microg/d dose groups was smaller than in the controls (P<.001). No significant difference was seen between the 0.05- microg/d dose group and the control group (P =.64). Survival for the 0.1- microg/d and 0.2- microg/d dose groups was lower than for the controls (95% in the controls vs 85.7% and 73.7%, respectively; P<.01). CONCLUSION: In the Tyr-Tag transgenic mouse, 1alpha-OH-D(2) inhibits pigmented ocular tumor growth at moderate drug levels with relatively low mortality. Clinical Relevance Vitamin D analogues merit further preclinical study in the treatment of ocular melanoma.

Effectiveness of vitamin D analogues in treating large tumors and during prolonged use in murine retinoblastoma models.

OBJECTIVE: To investigate the effectiveness of the vitamin D analogues 1,25-(OH)(2)-16-ene-23-yne vitamin D(3) (16,23-D(3)) and 1alpha-hydroxyvitamin D(2) (1alpha-OH-D(2)) in inhibiting retinoblastoma growth in large tumors in a xenograft model and with prolonged use in a transgenic model. METHODS: For the large-tumor study, the xenograft athymic mouse/human retinoblastoma cell (Y-79) model was used. Subcutaneous tumors were allowed to grow to an average volume of 1600 mm(3). Systemic treatment with 1 of the vitamin D analogues or with vehicle (control groups) was carried out for 5 weeks. For the long-term study, transgenic beta-luteinizing hormone-large T antigen (LHbeta-Tag) mice were systemically treated with 1 of the 2 compounds or vehicle (control groups) for up to 15 weeks. Tumor size and signs of toxicity were assessed. RESULTS: In the large-tumor study, tumor volume ratios for the 1alpha-OH-D(2) and 16,23-D(3) groups were significantly lower than those for controls (P<.002). No significant differences in tumor volume were seen between the 1alpha-OH-D(2) and 16,23-D(3) groups (P =.15). In the long-term study, the 1alpha-OH-D(2) group showed significantly smaller tumor size compared with its control (P<.001). No significant difference was seen between the 16,23-D(3) group and its control. Some toxic effects related to hypercalcemia were seen in both studies. CONCLUSIONS: In athymic mice in the large-tumor study, both 1alpha-OH-D(2) and 16,23-D(3) were effective in inhibiting tumor growth compared with controls. In the long-term study, 1alpha-OH-D(2) inhibited tumor growth but 16,23-D(3) did not. Effective doses of both compounds caused hypercalcemia and a significant increase in mortality. Clinical Relevance Use of 1alpha-OH-D(2) inhibited tumor growth in large tumors and with long-term treatment compared with controls. Because of hypercalcemia-related toxic effects seen in the present experiments, in clinical trials, serum calcium levels should be carefully monitored. This analogue may require use with drugs that lower serum calcium levels or use of relatively lower doses or skipped doses. The ideal alternative solution would be to identify vitamin D analogues that retain the antineoplastic action without the calcemic activity.

Phase II study of 1alpha-hydroxyvitamin D(2) in the treatment of advanced androgen-independent prostate cancer.

PURPOSE: In this single institution Phase II trial, we evaluated the efficacy of the vitamin D analogue, 1alpha-OH-D(2), in patients with advanced hormone-refractory prostate cancer. EXPERIMENTAL DESIGN: The patients initially received 1alpha-OH-D(2) at 12.5 micro g p.o. every day, which was dose adjusted for hypercalcemia. Given the cytostatic nature of the drug, the primary study end point was progression-free survival for a minimum of 6 months. The secondary end point was further characterization of drug toxicity. RESULTS: A total of 26 patients was enrolled. Using the intent-to-treat population, stable disease was seen for an average of 19.2 weeks (median 12 weeks, range 3-108 weeks). Twenty patients were evaluable for response. The one patient that achieved disease stabilization for >2 years elected to come off-study because of patient preference. His last disease evaluation showed no evidence of progression. No objective responses were seen. Previous and ongoing clinical observations strongly imply that PSA could be a misleading surrogate marker for clinical effect with this type of drug. Therefore, prostate-specific antigen was not used as a marker for disease response. Toxicity was as expected with mild hypercalcemia and associated symptoms like constipation and prerenal azotemia seen in some patients. Six (30%) evaluable patients experienced stable disease for >6 months, suggesting possible cytostatic activity. CONCLUSION: The results of this and other trials suggest further clinical investigation in this disease with vitamin D analogues alone or in combination with other agents, such as chemotherapy, should be pursued.

Toxicity and dose-response studies of 1-alpha hydroxyvitamin D2 in LH-beta-tag transgenic mice.

PURPOSE: To determine the effectiveness of a vitamin D analog, 1alpha-hydroxyvitamin D(2) (1alpha-OH-D(2)), in inhibiting retinoblastoma in a transgenic retinoblastoma model (LHbeta-Tag mouse) and to evaluate its toxicity. DESIGN: Experimental study using an animal (LHbeta-Tag transgenic mouse) randomized (controlled) trial. PARTICIPANTS AND CONTROLS: Two hundred seventeen LHbeta-Tag transgene-positive 8- to 10-week-old mice total; 179 drug-treated animals, 38 control animals. METHODS: Mice were fed a vitamin D- and calcium-restricted diet and were randomized to treatment groups receiving control (vehicle), or 0.1, 0.3, 0.5, or 1.0 micro g/day of 1alpha-OH-D(2) via oral gavage 5 times weekly for 5 weeks. Body weight was measured at the start of treatment and twice weekly during treatment. Animals were euthanized on the last day of treatment. The eyes were enucleated, processed histologically, and serially sectioned. Representative sections from the superior, middle, and inferior regions of each globe were examined microscopically and tumor areas were measured using Optimas software. Serum was collected for serum calcium levels. Kidneys were removed for histologic processing and were analyzed microscopically for kidney calcification. MAIN OUTCOME MEASURES: Mean tumor area was measured to determine drug effectiveness. Toxicity was assessed by survival, weight loss over the treatment period, serum calcium, and kidney calcification. RESULTS: The mean tumor size in each 1alpha-OH-D(2) group was smaller than controls (all P values < 0.02): control, 90,248 micro m(2); 0.1 micro g, 31,545 micro m(2); 0.3 micro g, 16,750 micro m(2); 0.5 micro g, 30,245 micro m(2); and 1.0 micro g, 16,049 micro m(2). No dose-dependent response curve was evident. The survival percentage for each group was as follows: control, 97%; 0.1 micro g, 91%; 0.3 micro g, 88%; 0.5 micro g, 70%; and 1.0 micro g, 63%. Mortality was higher in the 0.5- micro g and 1.0- micro g doses (P values < 0.01) compared with other treatment groups and with the control group. Serum calcium levels were significant in all treatment groups compared with controls (all P values < 0.0001). CONCLUSIONS: In the LHbeta-Tag mouse, 1alpha-OH-D(2) inhibits retinoblastoma with no significant increase in mortality in lower doses (0.1-0.3 micro g). 1alpha-OH-D(2) has approval by the Food and Drug Administration as an investigative drug for cancer treatment, and has shown efficacy with low toxicity in adult cancer trials. 1alpha-OH-D(2) meets the criteria for human clinical trials.

Toxicity and dose-response studies of 1alpha-hydroxyvitamin D2 in a retinoblastoma xenograft model.

BACKGROUND: Although calcitriol (1,25-dihydroxycholecalciferol) and vitamin D(2) inhibit retinoblastoma growth in the athymic (nude) mouse xenograft (Y-79 cell line) model of retinoblastoma, they can cause severe toxicity. OBJECTIVE: To examine the toxicity of and dose-dependent response for the inhibition of tumor growth for 1alpha-hydroxyvitamin D(2) (1alpha-OH-D(2)), an analogue with reduced systemic toxicity, in the athymic Y-79 mouse model. METHODS: Mice were randomized into treatment and control groups for 5-week toxicity and dose-response studies. Treatment was via oral gavage 5 times per week. Dose-response studies measured tumor inhibition and drug serum levels. Tumor size and body weight were measured weekly together with various criteria for toxicity. Animals were euthanized at the end of the treatment period. Tumors and kidneys were harvested, and serum was analyzed for calcium and drug levels. RESULTS: Doses of 0.1 to 1.2 microg/d were selected on the basis of toxicity studies for the dose-response trial. Tumor weight and volume in the 0.2-microg and 0.3-microg doses were significantly lower than in controls. Mortality rates and kidney calcification in mice treated with doses of 0.1 to 0.3 microg were lower than those observed in studies of calcitriol and vitamin D(2). CONCLUSION: A vitamin D analogue, 1alpha-OH-D(2), inhibits tumor growth in this xenograft model of retinoblastoma with less toxicity than calcitriol and vitamin D(2).

Toxicity and dose-response studies of 1 alpha-hydroxyvitamin D2 in LH beta-Tag transgenic mice.

PURPOSE: The study objective is to determine the effectiveness of a vitamin D analogue, 1 alpha-hydroxyvitamin D2 (1 alpha-OH-D2), in inhibiting retinoblastoma in a transgenic retinoblastoma model (LH beta-Tag mouse) and to evaluate its toxicity. Previous studies of 1 alpha-OH-D2 in athymic mice with human retinoblastoma xenografts suggested efficacy in tumor suppression and suitability for human treatment. METHODS: LH beta-Tag mice (N = 142), 8 to 10 weeks old, were randomly assigned to treatment groups receiving either control (vehicle) or 0.1, 0.3, 0.5, or 1.0 microgram/day of 1 alpha-OH-D2 via oral gavage five times a week for 5 weeks. Animals were then euthanized. The eyes were enucleated, processed histologically, and serially sectioned. Three sections of each eye were microscopically examined, and mean tumor area was measured using Optimus software. Toxicity was assessed by mortality, weight loss, serum calcium levels, and kidney calcification. RESULTS: The mean tumor size in each 1 alpha-OH-D2 group was smaller than in controls (P values < .02): control, 90,248 microns 2; 0.1 microgram, 31,545 microns 2; 0.3 microgram, 16,750 microns 2; 0.5 microgram, 30,245 microns 2; and 1.0 microgram, 16,049 microns 2. No dose-dependent response curve was evident. Mortality was higher in the groups receiving the 0.5 microgram and 1.0 microgram doses (P values < .01) than in the other treatment groups and the control group. CONCLUSION: In the LH beta-Tag mouse, 1 alpha-OH-D2 inhibits retinoblastoma with no increased mortality at lower doses (0.1 to 0.3 microgram). 1 alpha-OH-D2 has been approved by the Food and Drug Administration as an investigative drug for cancer treatment and has shown efficacy with low levels of toxicity in adult cancer trials. 1 alpha-OH-D2 meets the criteria for human clinical trials.

1alpha-hydroxyvitamin D2 is less toxic but not bone selective relative to 1alpha-hydroxyvitamin D3 in ovariectomized rats.

Identification of bone selective vitamin D analogues would provide an interesting substance class for the treatment of osteoporosis. The synthetic prodrug 1alpha-hydroxyvitamin D2 [1alpha(OH)D2] has been shown to combine equal bone-preserving activity with distinctly reduced calcemic effects relative to 1alpha-hydroxyvitamin D3 [1alpha(OH)D3] in 3-month-old ovariectomized (OVX) rats. Therefore, 1alpha(OH)D2 may be a bone-selective compound. The aim of this study was to compare the bone protective and the calcemic activities of chronically administered 1alpha(OH)D2 and 1alpha(OH)D3 in 6-month-old OVX rats over a broad dose range from ineffective to toxic doses. Ninety-six female 6-month-old Fischer-344 rats were used for this experiment. Eighty rats were bilaterally OVX, 8 rats were sham-operated (SHAM), and 8 rats were killed at the time of surgery as a baseline control. Groups of OVX rats received vehicle alone (n = 16) or daily doses in the diet of 0.025, 0.05, 0.1, and 0.2 microg of 1alpha(OH)D2 or 1alpha(OH)D3 per kg body weight (BW) per day (n = 8 each). After calcein double-labeling, all animals were killed 3 months post-OVX. Orally administered 1alpha(OH)D2 was significantly less toxic compared with 1alpha(OH)D3 in terms of BW gain and kidney calcium content. The effects of 1alpha(OH)D2 and 1alpha(OH)D3 on serum calcium and urinary calcium excretion were generally similar at all doses in this study. Both 1alpha(OH)D2 and 1alpha(OH)D3 prevented the estrogen deficiency-induced bone loss in OVX rats, and induced profound bone anabolic effects at high dosages. 1alpha(OH)D3 and 1alpha(OH)D2 also dose-dependently increased total bone mineral density (BMD), cortical area, and cortical thickness in the tibial diaphysis of OVX rats. Bone resorption as assessed by osteoclast numbers (Oc.Ns) in vertebral cancellous bone and urinary excretion of deoxypyridinoline (DPD) was dose-dependently suppressed by 1alpha(OH)D2 and 1alpha(OH)D3. These data show that although 1alpha(OH)D2 was slightly but significantly less toxic compared with 1alpha(OH)D3, it did not have increased skeletal effects at any dose. Taken together, our findings argue against selective metabolic activation of 1alpha(OH)D2 in bone.

Metabolism of [3alpha-3H] 25-hydroxyvitamin D2 in kidneys isolated from normal and vitamin D2-intoxicated rats.

With the availability of A-ring labelled 25OHD2, [3alpha-3H] 25OHD2, we have performed the present study to examine the metabolism of 25OHD2 using physiological substrate concentrations in perfused kidneys isolated from both normal and vitamin D2-intoxicated rats. Our results indicate that [3alpha-3H] 25OHD2 is metabolized into both 24(S),25,28-trihydroxyvitamin D2 [24(S),25,28(OH)3D2] and 24(R),25,26-trihydroxyvitamin D2 [24(R), 25,26(OH)3D2], and the amounts of these two metabolites produced in the kidney of vitamin D2-intoxicated rat were about 3-5 times higher than those produced in the kidney of normal rat. Similar results were also obtained with rat kidney homogenates incubated with [3alpha-3H] 25OHD2. Furthermore, we noted that the production of both 24(S),25,28(OH)3D2 and 24(R),25,26(OH)3D2 in the kidney homogenates of vitamin D2-intoxicated rats increased with the time of incubation and then subsequently decreased. The decrease in both 24(S),25,28(OH)3D2 and 24(R),25,26(OH)3D2 coincided with an increase in the fraction of total radioactivity distributed in the aqueous phase of the kidney homogenates. This finding suggested the possibility of further metabolism of 24(S),25,28(OH)3D2 and 24(R), 25,26(OH)3D2 into polar water-soluble metabolite(s). We then measured the radioactivity in the aqueous phase of kidney homogenates of both normal and vitamin D2-intoxicated rats incubated with [3alpha-3H] 25OHD2. It was noted that the amount of radioactivity in the aqueous phase of kidney homogenates of vitamin D2-intoxicated rats is higher than that present in the aqueous phase of kidney homogenates of normal rats. Thus, our study provides evidence for the first time for the formation of both 24(S),25,28(OH)3D2 and 24(R),25, 26(OH)3D2 under physiological conditions, and the possibility of their further metabolism into as yet unidentified polar water-soluble metabolite(s). As the formation of all these metabolites is increased in the kidney of vitamin D2-intoxicated rats when compared to normal rats, it appears that the increased rate of metabolism of 25OHD2 during hypervitaminosis D2 plays a significant role in the deactivation of 25OHD2.

New model of atherosclerosis in sand rats subjected to a high cholesterol diet and vitamin D2.

In order to defeat the atheroresistance in sand rats, 25 animals were given a high cholesterol diet for 45 days, which was then associated with oral treatment with vitamin D2 2000 IU/day for a further 45 days. At days 0, 45 and 90, plasma parameters, and aortic and heart morphology were examined. Results showed at D45 hypercholesterolaemia, increased plasma LDL and VLDL cholesterol, oxidized LDL, triglycerides, free fatty acids (FFA) and calcium levels and moderate hyperinsulinaemia. At D90, plasma-oxidized LDL and FFA were more enhanced, whereas calcium level was reduced. Development of hyperglycaemia was associated with hyperinsulinaemia and insulin resistance. The vitamin D2 administration induced advanced lesions, represented by the degenerescence of elastic lamina, smooth muscle cell proliferation and lipid calcic plaque at an ulcerated stage in most cases. The ischaemic effects were represented by acute myocardial infarction. The potential of the sand rat to develop atherosclerotic lesions at different stages opens the field to therapeutic tests of new anti-atherogenic agents.

[Activity of elastase and its inhibitors in arterial tissues and blood serum under experimental arterio-atherosclerosis]. / Aktyvnist' elastazy ta ïï inhibitoriv u tkanynakh arterii ta syrovatsi krovi za umov eksperymental'noho arterio-ateroskelrozu.

The role of the elastolytic system serum and arterial tissues in pathogenesis of vascular pathology was investigated on rabbits in experimental arterio-atherosclerosis. Activation of proteolytic enzymes, in particular elastase, sharp alteration capacity of inhibitors in arterial wall both at Menckeberg arteriosclerosis and atherosclerosis, permits to assume, that in the basis these different pathologic processes can lie general mechanisms, which will be realized through disturbance of the elastolytic system balance in blood vessels.

The novel analog 1,24(S)-dihydroxyvitamin D2 is as equipotent as 1,25-dihydroxyvitamin D3 in growth regulation of cancer cell lines.

The physiologically active metabolite of the vitamin D seco-steroid hormone, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), is a major regulator of mineral homeostasis. Recent evidence also suggests its role in regulating proliferation and differentiation of cells, including cancer cells. Therapeutic application of 1,25(OH)2D3 to hyperproliferative disease, such as cancer, is thwarted by its hypercalcemic activity. To overcome this problem, analogs of 1,25(OH)2D3 have been produced which retain growth regulating properties and exhibit decreased hypercalcemic activity. In the present study, the efficacy of the vitamin D2 analog, 1,24(S)-dihydroxyvitamin D2 (1,24(S)-(OH)2D2) in the inhibition of cancer cell proliferation and in inducing differentiation of cancer cells was compared to that of 1,25(OH)2D3. By the [3H]-thymidine incorporation procedure, 1,24(S)-(OH)2D2 is as equipotent as 1,25(OH)2D3 in inhibiting the proliferation of five different cell lines, ROS 17/2.8, the rat osteosarcoma cell line, MCF-7, the human breast cancer cell line, HD-11, the chick bone marrow v myc transformed cell line, HT-29, the human colon cancer cell line and HL-60, the human leukemia cell line. The inhibitory action was dose and time-dependent. The NBT reduction method indicated that 1,24(S)-(OH)2D2 induces the differentiation of the human leukemia cell (HL-60) to the same extent as 1,25(OH)2D3. Notwithstanding the vast similarity between 1,24(S)-(OH)2D2 and 1,25(OH)2D3 with regard to the above activities, they differ in their effects on calcium regulation. In conclusion, the present results encourage the use of 1,24(S)-(OH)2D2 for the treatment of cancer disease in vivo.

Comparison between dietary soybean protein and casein of the inhibiting effect on atherogenesis in the thoracic aorta of hypercholesterolemic (ExHC) rats treated with experimental hypervitamin D.

Atherosclerotic lesions of the thoracic aorta were induced in exogenously hypercholesterolemic (ExHC) rats by treating initially with hypervitamin D2 and subsequently feeding on hypercholesterolemic diets for 180 days. Dietary soybean protein, in comparison with casein, substantially decreased the degree of atherosclerotic lesions, which was evaluated by intimal thickening, although with a similar topographical distribution. The casein-fed rats tended to maintain a high concentration of serum cholesterol, particularly in triacylglycerol-rich lipoproteins. The concentrations of apo A-I and TBARS in the serum was comparable between the dietary protein groups. The data suggest that dietary soybean protein, compared to casein, produced lipoproteins which were less atherosclerotic by partitioning cholesterol in the triacylglycerol-poor lipoproteins.

A large dose of ergocalciferol does not cause deficient blood coagulation but is extremely toxic to rats.

Male Jcl:SD rats were fed vitamin D2 (ergocalciferol) at levels of 0 (control), 0.39, 0.63 and 1.00% or 0 (control), 0.0195, 0.0315 and 0.050% in the diet for 7 days. All rats of the 0.39-1.00% groups expired on days 2 and 3, while some rats of the 0.0195-0.050% groups died on days 3-6. LD50 (median lethal 7-day cumulative dose calculated from food intake) is 110.5 mg/kg (0.0354% dietarily). In expired and surviving treated rats, several organs (kidney, heart, etc.) were found to be mineralized; there were also renal tubular injuries and pulmonary bleeding. Centrilobular necrosis of liver was detected only in dead rats. Treatment also caused hypercalcemia but did not decrease blood coagulation factors. These results suggest that vitamin D does not have the effect of impeding blood coagulation but that it is extremely toxic, probably due to the hypercalcemia it causes.

Impairment of endothelium-dependent relaxation in aorta from rats with arteriosclerosis induced by excess vitamin D and a high-cholesterol diet.

The present investigation was undertaken to characterize the relaxing responsiveness in aortic strips from rats with arteriosclerosis, which was produced by excess vitamin D2 (VD) administration followed by treatment with or without a high-cholesterol diet for 6 weeks (VD + CHOL and VD group, respectively). This arteriosclerotic aorta was characterized by medial calcification and intimal cell proliferation. Helical strips of thoracic aorta were suspended in oxygenated Krebs-Henseleit solution. Under precontraction with noradrenaline, endothelium-dependent relaxations to acetylcholine were significantly attenuated in the VD and VD + CHOL as compared with the control. Relaxation to calcium ionophore A23187 was also significantly attenuated in the VD + CHOL. However, the relaxing responses to acetylcholine and A23187 in aortas from rats fed a high-cholesterol diet alone remained unaffected. Nitroglycerin caused an equal degree of relaxation in aortas from control and arteriosclerotic rats. There was a significant negative correlation between the relaxing response to acetylcholine and the calcium content in the aorta. These results indicate that in arteriosclerotic rat aortas, the endothelium-dependent relaxation to acetylcholine is impaired in proportion to the degree of calcification, and such impairment is facilitated by cholesterol feeding but can not be attributed to hypercholesterolemia or vascular cholesterol deposition.

Protective effects of benidipine hydrochloride (KW-3049), a calcium antagonist, against experimental arterial calcinosis and endothelial dysfunction in rats.

Protective effects of benidipine hydrochloride (KW-3049) against arterial calcinosis and its possible mechanisms of action have been investigated. Arterial calcinosis was induced in rats by combined administration of vitamin D2 (1050000 IU/kg, s.c.) and nicotine (12.5 mg/kg, p.o., b.i.d.) for 6 successive days. Calcium antagonists, benidipine or nifedipine, were given orally twice a day during the same period. The aortic calcium content in vitamin D2 and nicotine-treated (control) rats increased to about 25 times that in normal rats, accompanying an increase of serum calcium level. Benidipine (10 mg/kg, p.o., b.i.d.) reduced the aortic calcium content to about 18% of control rats without reducing the serum calcium level. Although the presence of aortic endothelial cells was observed under light microscopy in control rats, their surfaces were degenerated under scanning electron microscopy. Benidipine exerted a protective effect against these degenerative changes. Acetylcholine-induced endothelial dependent relaxation was attenuated in control rats, compared with that in normal rats. Benidipine significantly improved this attenuation of the relaxation. These results suggest that the anticalcinotic effect of benidipine is accompanied by its protective effect on endothelial cells.