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A 76-year-old female patient who had been taking vitamin D2 100,000 U/day for more than 14 years due to hypoparathyroidism following total throidectomy was admitted because of protracted hypercalcemia. On admission, the levels of serum vitamin D2 (99.8 ng/ml) and 25-OHD2 (356 ng/ml) were very high, and 1,25-(OH)2D2 was low (4.0-18.7 pg/ml). Serum D3' 25-OHD3 and 1,25-(OH)2D3 were below the normal range. Despite intensive hydration with saline, intravenous hyperalimentation with phosphate- and calcium-free nutrients, and administration of glucocorticoid and calcitonin, the hypercalcemia persisted, accompanied by hypoproteinemia, edema, pleural effusion and congestive heart failure. The serum D2 and 25-OHD2 concentrations remained high and were accompanied by a gradual increase in 1,25-(OH)2D2 (121 pg/ml), which further increased after the administration of bisphosphonate (pamidronate) to 183 pg/ml. Seventeen months later, serum calcium and 1,25-(OH)2D2 were normalized but serum D2 and 25-OHD2 remained high. The serum 24,25-(OH)2D2/25-OHD2 ratio was relatively constant throughout her clinical course, whereas the low serum 1,25-(OH)2D2/25-OHD2 ratio at admission gradually increased during admission, suggesting that the increase in serum 1,25-(OH)2D2 is due to increased production rather than decreased degradation. The administration of pamidronate further increased serum 1,25-(OH)2D2. These features of the clinical course demonstrate that the 1,25-dihydroxyvitamin D concentration in hypercalcemic patients with protracted vitamin D intoxication may be decreased, normal or increased. Possible factors responsible for a protracted increase in serum 1,25-(OH)2D2 are body weight loss, hypoproteinemia, and phosphate depletion. In addition, some bisphosphonates would certainly promote PTH-independent production of 1,25-(OH)2D2.
Vitamins D2, D3 and E were resolved and quantified by applying reversed-phase high-performance liquid chromatography to extracts of cod liver oil. The method, using two reversed-phase C18 columns and a ternary mixture of acetonitrile, methanol and water as the eluent resolved all fat-soluble vitamins well, including the pair D2-D3. The extraction procedure was studied; the recoveries, using two different solvents (hexane and diethyl ether) for extractions were 60.6 +/- 1.0 and 77.1 +/- 1.1, 56.9 +/- 1.2 and 74.8 +/- 0.8, and 14.1 +/- 0.7 and 89.8 +/- 1.4% for vitamins D2, D3 and E, respectively.
Serum concentrations of 25-hydroxyvitamin D (25-OHD) and 1,25-dihydroxyvitamin D [1,25-(OH)2D] of vitamin D2 and D3 origin were determined separately in 10 women before vitamin intake in early pregnancy, and repeated in maternal and cord serum obtained at delivery after 20 to 30 wk of vitamin D2 supplementation in a dose of 400 IU/day. Before supplementation 25-OHD2 and 1,25-(OH)D2D2 were present in just traceable or nondetectable concentrations, but the levels increased in all to a mean +/- 1 SD of 7.3 +/- 3.7 ng/ml and 37.2 +/- 18.1 pg/ml, respectively (p less than 0.0025), by the time of delivery. At delivery the total 25-OHD and 1,25-(OH)2D levels were always lower in the cord than in the maternal serum (30.7 +/- 14.2 versus 20.1 +/- 9.1 ng/ml, and 90.1 +/- 31.2 versus 37.3 +/- 11.6 pg/ml, p less than 0.0025). The paired concentrations of 25-OHD were closely related (r = 0.89, p less than 0.0005), while the association for 1,25-(OH)2D was not statistically significant (r = 0.53, p less than .01). The 25-OHD of D2 and D3 origin accounted for a similar proportion of the total 25-OHD in the maternal and cord serum (ratio of 25-OHD2 to 25-OHD3: 0.40 +/- 0.28 versus 0.45 +/- 0.29, p = NS), as did the respective 1,25-(OH)2D metabolites [ratio of 1,25-(OH)2D2 to 1,25-(OH)2D3: 0.73 +/- 0.35 versus 0.90 +/- 0.50, p = NS].(ABSTRACT TRUNCATED AT 250 WORDS)
A method has been optimized for the conversion of ergosterol in mushrooms to vitamin D2, and the vitamin D-enriched mushrooms have been tested for bioavailability of vitamin D2 using a rat model. Femur bone mineral density (BMD) of the experimental group of animals fed with vitamin D2 (1 microg/d) obtained from irradiated mushrooms was significantly increased. Femur BMD of two groups was significantly higher. Femur BMD of the experimental group was significantly elevated compared to initial femur BMD of the study group. Data indicate that vitamin D2 from ultraviolet (UV)-irradiated mushrooms was well absorbed and metabolized in animals.
In the four-week study the antirachitic action of vitamin D2 in different does (5000, 7500, 10 000 I. U. per kg food) and of vitamin D3 in a dose of 500 I. U. per kg food was investigated in chicks. The vitamin efficiency was measured with respect to the following seven parameters: weight gain, serum content of Ca and P, serum activity of alkaline phosphatase, bone ash content, content of Ca-binding protein in the duodenal mucosa, and weight of parathyroid glands. In regard to these parameters, vitamin D2 in a dose of 7500 I. U./kg and vitamin D3 in a dose of 500 I.U./kg produced a similar effect. Thus, the antirachitic effect of vitamin D3 was 15 times higher than that of vitamin D2. In the three-day study of the effect of vitamins D2 and D3 on the synthesis of Ca-binding protein it was found that they exerted a similar effect when the dose of vitamin D2 was 14.5 times higher than that of vitamin D3.
I describe a new assay that is capable of measuring vitamin D2, vitamin D3, 25-hydroxyvitamin D2, and 25-hydroxyvitamin D3 in 2 ml of plasma or serum. Plasma is extracted by the Bligh and Dyer technique [Can. J. Biochem. Physiol. 37, 911 (1959)], the lipid component is fractionated by two high-performance liquid-chromatographic systems based upon adsorption and reversed-phase chromatography, and each of the four vitamin D metabolites is measured by its absorbance at 254 nm. The method has a sensitivity limit of 0.5 mug/liter of plasma. The identity of metabolite peaks was confirmed by mass spectrometry, ultraviolet absorption spectrophotometry, and rechromatography, and there was good correlation (r=0.84) between plasma 25-hydroxyvitamin D as measured by the present method and by a protein binding assay developed in our laboratory. Mean concentrations of vitamin D and 25-hydroxyvitamin D in normal adults (n=25) in December were 2.2 +/- 1.1 (SD) and 16 +/- 3.9 (SD) mug/liter, respectively. 25-Hyroxyvitamin D2 made up 31% of the total 25-hydroxyvitamin D. Patients receiving pharmacological doses of vitamin D had values for vitamin D and 25-hydroxyvitamin D that were 10- to 100-fold normal. This method provides a rapid, reliable physico-chemical assay that appears to have advantages over existing protein binding assays and can be used to measure circulating vitamin D.
A semi-automatic procedure for the continuous clean-up and concentration of several fat-soluble vitamins prior to their separation by HPLC and UV detection is reported. The procedure is based on the use of a minicolumn packed with aminopropylsilica as sorbent located prior to the chromatographic detection system. The overall process was developed and applied to the main liposoluble vitamins (A, D2, D3, E, K1, K3) and several hydroxy metabolites of vitamin D3 [25-(OH)-D3,24,25-(OH)2-D3 and 1,25-(OH)2-D3]. All the analytes were monitored at a compromise wavelength of 270 nm. Calibration graphs were constructed between 0.01 and 100 ng ml-1 for vitamin D2 and D3 and their hydroxy metabolites, between 0.1 and 100 ng ml-1 for vitamin A, K1 and K3 and between 1 and 100 ng ml-1 for vitamin E, with excellent regression coefficients (> or = 0.9901) in all cases. The precision was established at two concentration levels with acceptable RSDs in all instances (between 3.6 and 8.7%). The method was appropriate for the determination of vitamin D2, D3, K1 and K3 and the 24,25-dihydroxy and 25-hydroxy metabolites of vitamin D3 in human plasma. The method was applied to plasma samples spiked with the target analytes and the recoveries ranged between 78 and 109%.
A radio-immunoassay (RIA) for determination of 1,25-dihydroxycholecalciferol (1,25(OH)2D3) and 1,25-dihydroxyergocalciferol (1,25(OH)2D2) in serum was compared with a competitive protein binding assay using calf thymus receptor to clarify the comparability of measurements in subjects treated with either vitamin D2 or vitamin D3 (4000 IU per day for 8 weeks). Before and during treatment with vitamin D3 the two assay techniques were concordant, but during vitamin D2 treatment the serum concentrations of 1,25(OH)2D2 and 1,25(OH)2D3 were higher when measured by RIA. The study demonstrates that during vitamin D2 treatment unknown compounds, recognized only by the antiserum, co-elute with 1,25(OH)2D2 and 1,25(OH)2D3 in high pressure liquid chromatography. It can be concluded that the radio-immunoassay used here cannot replace the calf thymus receptor assay.
Vitamin D2 and D3 intake and plasma 25OHD2 and 25OHD3 were measured in 70 elderly women; 13 living at home and 57 long-stay patients with no access to sunlight. Vitamin D2 intake and plasma 25OHD2 were correlated in the whole group (p less than .005) and vitamin D3 intake and plasma 25OHD3 and total D intake and total 25OHD were significantly correlated (p less than .005) in the patients. In the whole group the plasma 25OHD2 increased by 4.5 nmol/l for every 1 microgram increase in vitamin D2 intake. This was also the increase observed in a longitudinal study of vitamin D2 supplements in 11 patients. Vitamin D intake is a significant determinant of plasma 25OHD and the relation between them suggests that stores of vitamin D can be maintained at 20 nmol/l in the elderly by a daily intake of 4 micrograms of vitamin D, even in the absence of sunlight.
A single column reverse phase high pressure liquid chromatographic method is described for the determination of vitamins D2 and D3 in fluid milk. Resolution of vitamin D2 from D3 is helpful for use as an internal standard. The method involves overnight saponification at room temperature, extraction of unsaponifiables, precipitation of cholesterol, and aluminum oxide column cleanup. Sample extracts were chromatographed under isocratic conditions on a 10 micron Vydac reverse phase column using acetonitrile-methanol (90 + 10) as the mobile phase. In addition, a MicroPak MCH-5 reverse phase column with acetonitrile as the mobile phase was used with an automatic system for one product type. Thirty samples each of homogenized (3.8% fat), low fat (2.0% fat), and skim (less than or equal to 0.5% fat) milk spiked with 200, 400, and 600 IU vitamin D/qt were analyzed. Coefficient of variation (CV) and percent recovery for each product type and each spike level of vitamins D2 and D3 were calculated from 10 replicate analyses. Vitamin D2 recoveries for all product types at the 3 fortification levels varied from 85.2 to 99.7%; vitamin D3 recoveries varied from 85.9 to 98.8%. The minimum detectable quantity of vitamin D in milk was 15 IU/qt.
In vitro hydroxylation of vitamin D2 at carbon-24 (C-24) was demonstrated with pig liver homogenate. The putative 24-hydroxyvitamin D2 (24-OHD2) comigrated with standard 24-OHD2 on a Zorbax Sil column developed in hexane/isopropanol (98/2). Rechromatography in methylene chloride/methanol (99.8/0.2) resolved the putative 24-OHD2 into two components. The identity of these compounds was determined to be 24(R)-OHD2 and 24(S)-OHD2 (epimers) by low resolution mass spectroscopy and proton NMR spectroscopy. The fact that epimers of 24-OHD2 were produced from vitamin D2 in the absence of pig liver homogenate in vitro was strong evidence for the participation of free radicals in the reaction. Further support for free radical involvement was provided by the following observations: (a) hydroxyl free radical scavengers such as alpha-tocopherol, catalase, and ethanol reduced the amount of 24-OHD2 produced by 18-64%; (b) use of autoclaved homogenate in the incubation mixture had little or no effect on the amount of 24-OHD2 produced; and (c) the failure of the enzyme-substrate saturation curve to level off as expected with high levels of vitamin D2 (100-2000 micrograms = 50-1009 microM). Maximum production of 24-OHD2 was obtained at pH 4.75 and represented a sevenfold increase relative to the amount produced at pH 7.4. The omission of citrate or the addition of electron transport inhibitors, cyanide or antimycin, had little or no effect on the reaction. These data suggested that C-24 hydroxylation of vitamin D2 in vitro was a free radical-mediated process not involving the electron transport system. In vitro hydroxylation at C-24 appeared to be driven by free radicals, and the dominance of this reaction made it difficult to determine whether there was an enzyme involved in the reaction.
A liquid chromatographic (LC) method was developed for determining vitamins D2 and D3 in fortified milk and infant formulas. The lipid-soluble components were extracted from the aqueous phase by homogenizing in isopropanol-methylene chloride with magnesium sulfate added to remove water. The vitamins were fractionated from the lipid material by using gel permeation chromatography (GPC) followed by further cleanup of the combined GPC fractions on a muBondapak/NH2 column. Four muStyragel (100 A) columns connected in series were used for GPC fractionation of sample extracts in methylene chloride. Injection and collection were repeated 3 times to collect enough vitamin D for quantitation. The muBondapak/NH2 column, using a mobile phase of methylene chloride-isooctane-isopropanol (600 + 400 + 1), resolved vitamin D from other UV-absorbing compounds and soy sterols in infant formula and from cholesterol in milk. Vitamins D2 and D3 coeluted as one peak, with the resolution and vitamin level sufficient for visual monitoring (280 nm/0.02 absorbance unit full scale) in a collection time of 22-26 min. A Zorbax ODS (6 micron) column and a methylene chloride-acetonitrile-methanol (300 + 700 + 2) mobile phase were used for LC quantitation; vitamins D2 and D3 were baseline resolved in about 11 min. The infant formula samples included ready-to-use and concentrated liquids prepared in nonfat milk base or soy base fortified with vitamins D2 or D3 at 400 IU/qt or L (10 micrograms). The mean percent recovery of added vitamin D3 (400-500 IU/qt) from infant formula (n = 7) was 89.6 +/- 6.7 (coefficient of variation (CV) 7.5%).(ABSTRACT TRUNCATED AT 250 WORDS)
The use of Fourier transform 1H NMR to characterize vitamin D2 metabolites is described. A 300-MHz spectrometer capable of generating a 6-microseconds pulse and a sweep width of 4000 Hz was used. High-resolution spectra were obtained on 5 micrograms of material using standard 5-mm NMR tubes fitted with glass inserts and isotopically enriched chloroform-d solvent. The data acquisition time under these conditions was 4 h. Application of this technique to a variety of both synthetic and naturally occurring vitamin D2 metabolites, in addition to synthetic delta 22-1,25-dihydroxyvitamin D3, resulted in the reassignment of the chemical shifts for the C-21 and C-28 methyl groups of vitamin D2. The C-21 methyl group resonance is now assigned to the doublet appearing at delta 1.01, whereas the C-28 signal corresponds to the doublet at delta 0.90. An examination of the spectrum of 24 (R),25-dihydroxyvitamin D2 also led to the reassignment of the side-chain methyl group resonances. This technique is an additional means of identifying microgram quantities of vitamin D metabolites.
The present investigation was directed towards finding the relative biopotency of vitamin D3 and D2 in fish. The freshwater column feeder fish Labeo rohita (Rora) was used for the study. The feeding of Rora with graded levels of vitamin D2 (550, 1,100 and 1,650 i.u./kg diet) and vitamin D3 (1,100 and 1,650 i.u./kg diet) resulted in no behavioural or morphological changes in comparison with the group fed a vitamin D-deficient diet. Also, the growth rate, feed efficiency, mortality rate, carcass protein, total lipids, calcium and phosphorus were found to remain unaltered in the vitamin D-deficient fish and fish fed any form of the vitamin. Further, there is no difference in any of the above parameters between the different doses of vitamin D3 or vitamin D2. Thus, the results of this study indicate that both of the forms of vitamin D (D2 or D3) are not biologically active for Rora (Labeo rohita) as a representative of freshwater fish.
A metabolite of vitamin D2 in the tissues of primates fed this vitamin co-migrates to a significant extent with 24,25-dihydroxyvitamin-D3 on columns of Sephadex LH20, with chloroform/n-hexane (60/40 by vol) as eluent. In individuals receiving vitamin D2 and in individuals with scant exposure to sunlight this metabolite, which we believe to be 25,26-dihydroxyvitamin-D2, may yield falsely higher concentrations of apparent 24,25-dihydroxyvitamin-D3 if separation on LH20 is used.
The objective of this study was to investigate the effects of inositol and vitamin D2 on bone uptake of 45Ca in rats. The radioactive calcium was administered to young rats by orogastric intubation (2 muci/100 g body weight (b. wt)) with inositol (20 mg/100 g b. wt.) and/or vitamin D2 (5OO IU/100 g b. wt.) to normal rats. Bone uptake of 45Ca was measured after 24 hours by standard technique. Inositol alone produced a 48% increase in calcium uptake. It is concluded that inositol significantly increases bone uptake of radioactive calcium (P greater than 0.005). Simultaneous administration of vitamin D2 decreases the effect of inositol considerably, while vitamin D2 has no significant effect.