Vitamin D supplements and 25-hydroxy vitamin D concentrations in the elderly.

Serial 25-hydroxy vitamin D (25-OHD) concentrations were measured in long-stay geriatric patients treated with vitamin D. Comparison between a treatment and a control group showed that a daily dose of 500 IU vitamin D produced a significant increase in 25-OHD levels by two months. The supplement had a striking effect when the initial 25-OHD level was low and very little effect when it was high. 25-OHD levels in subjects on 2000 IU vitamin D daily were only marginally higher than those in subjects on 500 IU. A dose of 500 IU vitamin D daily should therefore produce adequate blood 25-OHD concentrations in most old people, and probably prevent most cases of osteomalacia in the elderly--though a large-scale study is needed to confirm this.     

Vitamin D status in different subgroups of British Asians.

To assess the effect of religious dietary practices and social customs on the vitamin D status of Asian immigrants, we kept records of the dietary intake and time spent out of doors of 81 Ugandan Asian men, women, and girls (9-19 years old). Sera were analysed for 25-hydroxycholecalciferol (25-OHD3), and 28% of the subjects were found to have levels below the lower limit of normal. The (vegetarian) Hindus had the lowest dietary intakes, least time out of doors, and lowest serum 25-OHD3. The Goan (Roman Catholic) Asians, despite more pigmentation, had 25-OHD3 levels similar to those found among indigenous British people and had the most satisfactory vitamin D intakes. Among Asians, whose exposure to sunlight may be limited, dietary vitamin D becomes the major determinant of serum 25-OHD3.     

Diet,sunlight, and 25-hydroxy vitamin D in healthy children and adults.

In 110 white West Midlands children serum 25-hydroxy vitamin D (25-OHD) concentrations showed a pronounced seasonal variation, the values being highest in August and lowest in February. The concentrations correlated significantly both with recorded sunlight and with seasonal ultraviolet energy of the sunlight. Children who had had a seaside holiday the previous summer had a higher mean 25-OHD concentration than those who had not had a summer holiday away from home. Correlation between vitamin D intake and serum 25-OHD concentration was not significant.     

Ethnic-specific differences in vitamin d status is associated with adiposity

Background

Low circulating 25 hydroxyvitamin D [25(OH)D] concentrations are common in obesity (BMI ≥30 kg/m²) and a negative relationship with body fat distribution has recently been reported. Ethnic-specific differences in body fat distribution have been described with South Asians are reported to have greater visceral adipose tissue (VAT), which could influence circulating 25(OH)D concentrations. The objective of this study is to investigate the relationship between plasma 25(OH)D, adiposity, and body fat distribution in Europeans and South Asians.

Methods/Principal Findings

187 Europeans and 192 South Asians were assessed for demographics, anthropometrics, and plasma 25(OH)D concentrations. Subcutaneous adipose tissue (SAT) and VAT were quantified by CT scan, and percent body fat by DEXA. Data were assessed by general linear models. South Asians had lower (P<0.001) plasma 25(OH)D concentrations and higher VAT (P = 0.04) than Europeans. Plasma 25(OH)D concentrations were negatively (P<0.05) associated with BMI, waist circumference, percent body fat, total adipose tissue, VAT, and SAT in unadjusted models and negatively (P<0.05) associated with VAT, SAT, and percent body fat after adjusting for BMI, ethnicity, age, and season of blood collection in males and females. When percent body fat, VAT, and SAT were included in the same model, only VAT remained negatively (P<0.05) associated with plasma 25(OH)D concentrations. Ethnicity remained significant in all models (P<0.001).

Conclusion

Compared to other adipose tissue compartments, VAT may have a distinct role in determining plasma 25(OH)D concentrations, which may account for the lower levels in South Asians.     

The quantitative determination of vitamin D3 and its metabolites in plasma

A method is described which enables determination of vitamin D3 and its physiologically most important metabolites, i.e. 25-OHD3, 24,25-(OH)2D3, 25,26-(OH)2D3 and 1,25-(OH)2D3 in a plasma sample of about 2 to 4 ml. The whole procedure involves two preparative and one analytical steps: Extraction with methanol/methylene chloride (2:1), chromatographic separation on Lipidex 5000 using a stepwise gradient of n-hexane and chloroform and finally HPLC separation on Zorbax-Sil columns with n-hexane isopropanol mixtures and subsequently reversed phase separation on RP 18-columns and mixtures of methanol and water. Except for 1,25-(OH)2D3 all D compounds were quantified by UV-detection with 1.4 ng of substance being the lowest detectable amount. 1,25-(OH)2D3 was measured by radioimmunoassay. Prior to HPLC analysis the extract was separated into three fractions on Lipidex 5000 which contained 1) vitamin D3, 2) 25-OHD3 and 3) the dihydroxy metabolites. The three fractions were separated by HPLC using different mixtures of isopropanol/n-hexane and methanol/water, respectively. Retention times of the individual D-components longer than 10 min appeared to be essential to separate these compounds from accompanying material. Overall recoveries of the individual metabolites were for vitamin D3 48.9%, for 25-OHD3 54.2%, for 24,25-(OH)2D3 50.9% and for 1,25-(OH)2D3 52.5%. Application of the methods to plasma samples from pigs with pseudovitamin D deficiency rickets, typ I, revealed a reduced concentration of 1,25-(OH)2D3 and 24,25-(OH)2D3 and an elevated level of 25-OHD3 in these animals. The results obtained by this method contributed substantially to a better understanding of the aetiological factors associated with this disease.     

Vitamin D is a major determinant of bone mineral density at school age

Background

Vitamin D insufficiency in children may have long-term skeletal consequences as vitamin D affects calcium absorption, bone mineralization and bone mass attainment.

Methodology/Principal Findings

This school-based study investigated vitamin D status and its association with vitamin D intake and bone health in 195 Finnish children and adolescents (age range 7–19 years). Clinical characteristics, physical activity and dietary vitamin D intake were evaluated. Blood and urine samples were collected for serum 25-hydroxyvitamin D (25-OHD) and other parameters of calcium homeostasis. Bone mineral density (BMD) and body composition were measured with dual-energy X-ray absorptiometry (DXA). Altogether 71% of the subjects were vitamin D insufficient (25-OHD <50 nmol/L). The median 25-OHD was 41 nmol/L for girls and 45 nmol/L for boys, and the respective median vitamin D intakes 9.1 µg/day and 10 µg/day. In regression analysis, after adjusting for relevant factors, 25-OHD concentration explained 5.6% of the variance in lumbar BMD; 25-OHD and exercise together explained 7.6% of the variance in total hip BMD and 17% of the variance in whole body BMD. S-25-OHD was an independent determinant of lumbar spine and whole body BMD and in magnitude surpassed the effects of physical activity.

Conclusions/Significance

Vitamin D insufficiency was common even when vitamin D intake exceeded the recommended daily intake. Vitamin D status was a key determinant of BMD. The findings suggest urgent need to increase vitamin D intake to optimize bone health in children.     

Effect of phototherapy on serum 25-hydroxyvitamin D in the Antarctic

The hypothesis that sunlight may induce the enzymes involved in the vitamin D pathway has been tested by comparing the ability to synthesize vitamin D3 and its 25 hydroxy metabolite (25-OHD) in 2 groups of male volunteers resident at the British Antarctic base at Rothera Point (67 degrees 34'S.). One group endured the UV depleted winter and the other group received regular phototherapy throughout the winter. Both groups then received a course of 14 days phototherapy in October (Southern Hemisphere spring). The group receiving regular phototherapy had a trend towards a higher level of serum 25-OHD, and the October phototherapy course produced a further small increase in serum 25-OHD values. In the previously non irradiated group the October phototherapy produced a much larger increase in serum 25-OHD so that they attained the previously higher values of the pre-iradiated group. There was a negative correlation between the pre October phototherapy serum concentration of 25-OHD and the subsequent increment (r-0.78, p less than 0.01) but no relationship between the serum 25-OHD and D3 after phototherapy. These results provide evidence against the existence of enzyme induction of vitamin D 25 hydroxylase by light.     

Actions of vitamins D2 and D3 and 25-OHD3 in anticonvulsant osteomalacia.

In 54 epileptic outpatients treated for at least one year with anticonvulsants the bone mineral content (B.M.C.), an estimate of total body calcium, and serum calcium were measured before and during treatment with three doses of cholecalciferol (vitamin D3; 200, 100, and 50 mu-g daily) and 25-hydroxycholecalciferol (25-OHD3; 40, 20, and 10 mu-g daily) for 12 weeks. The results, when compared with the effects of calciferol (vitamin D2; 200, 100, and 50 mu-g daily) in 40 epileptic outpatients, showed different actions in anticonvulsant osteomalacia of vitamin D2 on the one hand and vitamin D3 and 25-OHD3 on the other. In the patients who received vitamin D2 an increase in B.M.C. was found whereas serum calcium was unchanged. The patients who received vitamin D3 or 25-OHD3 showed an increase in serum calcium but unchanged values of B.M.C. The results suggest that liver enzyme induction cannot alone explain anticonvulsant osteomalacia.     

Vitamin D state of Asians living in Pakistan.

Asians living in Pakistan have serum 25-hydroxycholecalciferol concentrations which are well within the normal range and significantly higher than in Asians resident in Britain. Clearly, Asians can maintain an adequate vitamin D state given an abundant supply of ultraviolet light, and the expression of vitamin D deficiency is an environmental and not a genetically determined characteristic. The risk of an Asian developing vitamin D deficiency increases after migration to Britain. The most efficient way to treat this is by supplementation with vitamin D.     

The biological assessment of vitamin D3 metabolites produced by rumen bacteria

Biological assays were performed to evaluate 10-oxo-19-nor-vitamin D3 (10-oxo-D3) and 5(E) 25-hydroxy-10-oxo-19-nor-vitamin D3 (25-OH-10-oxo-D3) two bacterial products of vitamin D3 (D3) and 25-hydroxyvitamin D3 (25-OHD3) metabolism, respectively. The 5(Z) and 5(E) isomers of 10-oxo-D3 were, respectively, 40- and 80-fold less active than D3 in stimulating Ca+2 absorption from the gut. 25-Hydroxy-10-oxo-D3 did not stimulate Ca+2 absorption. Only 5(Z) 10-oxo-D3 induced mobilization of bone Ca+2. In addition, both 10-oxo-D3 and 25-OH-10-oxo-D3 showed poor affinities for either the plasma D3-binding protein or the thymus 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] receptor. 10-Keto-D3 exhibited a plasma half-life of only 6 min. This was a much shorter half-life than that exhibited by other vitamin D metabolites and was expected because of the poor affinity 10-oxo-D3 has for the plasma vitamin D binding protein. Bacterial metabolism of D3 deactivates the vitamin, which allows ruminants to tolerate relatively large oral doses of D3.     

The role of vitamin E in metabolism and reception of vitamin D

It was found that calcium exchange disturbances under vitamin E deficiency is due to changes in the metabolism of vitamin D. In vitamin E-deficient rats the serum blood levels of hydroxyvitamin D (25-OHD) showed no significant changes, whereas the concentration of the hormonal form of 1.25-hydroxyvitamin D [1.25(OH)2D], decreased by 40%. In vitro studies showed that the 25-hydroxylase D3 activity in the livers of rats with E-avitaminosis had a tendency to decrease (by 22%), whereas that of 24-hydroxylase dropped drastically (by 52%). The serum blood levels of the parathyroid hormone (PTH) and kidney levels of cAMP under E-avitaminosis were significantly lowered. Preincubation of kidney slices with the adenylate cyclase activator, forskolin, increased the activity of 1-OHase in about the same degree as that in vitamin E-rich rats. The free radical scavenger, BHT, added to kidney slices suppressed the activity of the both enzymes; this finding testifies to the low O2-binding affinity of these monooxygenases. The content of 1.25(OH)2D3 receptors occupied in vivo in the kidneys of vitamin E-deficient rats decreased 2.5-fold; however, the binding of 1.25(OH)2D3-receptor complexes to heterologous DNA was unaffected thereby. The vitamin deficiency in vivo results in the inhibition of vitamin D metabolism in the liver and kidney concomitant with the formation of active metabolites and decreases the concentration of hormone-receptor complexes in target tissues.     

Seasonal fluctuations in serum concentrations of vitamin D metabolites in normal subjects.

Serum concentrations of 25-hydroxycholecalciferol (25-OHD), 24,25-dihydroxycholecalciferol (24,25-(OH)2D), and 1,25-dihydroxycholecalciferol (1,25-(OH)2D) were measured at monthly intervals throughout the year in eight normal subjects. 25-OHD was measured by competitive protein-binding assay after Sephadex LH 20 chromatography, 24,25-(OH)2D by competitive protein-binding assay after Sephadex LH 20 and high-pressure chromatography, and 1,25-(OH)2D by radioimmunoassay after the same separation procedure as for 24,25-(OH)2D. A seasonal variation, apparently dependent on exposure to ultraviolet light, was found for all three metabolites. A study in six other normal subjects showed that there was no diurnal rhythm in any of the metabolites. Oral administration of 2 microgram 1,25-(OH)2D caused a sharp rise in serum concentrations of 1,25-(OH)2D and no change in the concentrations of the two other metabolites, but by 12 hours the 1,25-(OH)2D concentration had returned to the basal value. The concentrations of all three metabolites studied vary according to the season. Thus to interpret these concentrations in any subject the normal range for the particular season must be referred to.     

25-Hydroxycholecalciferol absorption in steatorrhoea and postgastrectomy osteomalacia.

Post-absorption levels of 25-hydroxy vitamin D (25-OHD) after oral administration of 25-hydroxycholecalciferol (25-OHD3) were measured in 11 subjects. Five had presented with steatorrhoea of various causes while six had post-gastrectomy osteomalacia. Post-absorption levels of 25-OHD were low in four of the patients with steatorrhoea but normal in five of those with post-gastrectomy osteomalacia. There was a significant inverse correlation between peak post-absorption 25-OHD levels and faecal fat excretion. All patients with active post-gastrectomy osteomalacia had subnormal baseline plasma 25-OHD levels, which indicates that the condition is due to a deficiency of vitamin D. Only two of the patients with osteomalacia had estimated dietary vitamin D intakes ofer 1-75 microng/day. These findings suggest that an oral 25-OHD absorption test may be a valuable measure of small intestinal function and that poor dietary vitamin D intake rather than impaired absorption of the vitamin may be the major cause of post-gastrectomy osteomalacia.     

Evaluation of Vitamin D Repletion Regimens to Correct Vitamin D Status in Adults

ObjectiveTo determine the efficacy and safety of commonly prescribed regimens for the treatment of vitamin D insufficiency.MethodsWe performed a retrospective analysis of 306 consecutive patients who were prescribed ergocalciferol (vitamin D₂) for correction of vitamin D insufficiency at the Atlanta Veterans Affairs Medical Center between February 2003 and May 2006. Serum levels of parathyroid hormone, 25-hydroxyvitamin D (25-OHD), and calcium were compared before and after treatment with ergocalciferol. Patients who did not have a 25-OHD determination (n = 41) were excluded from analysis. Vitamin D deficiency, insufficiency, and sufficiency were defined as a serum 25-OHD level of < 20 ng/mL, 21 to 29 ng/mL, and > 30 ng/mL, respectively.ResultsWe identified 36 discrete prescribing regimens. The 3 most common regimens were ergocalciferol 50,000 IU once weekly for 4 weeks followed by 50,000 IU once monthly for 5 months (n = 48); ergocalciferol 50,000 IU once monthly for 6 months (n = 80); and ergocalciferol 50,000 IU 3 times weekly for 6 weeks (n = 27). Each of these 3 treatments significantly increased serum 25-OHD (P < .01), but vitamin D sufficiency was achieved in only 38%, 42%, and 82% of study subjects, respectively. Regimens with > 600,000 IU of ergocalciferol given for a mean of 60 ± 40 days achieved sufficiency in 64% of cases, without vitamin D toxicity.ConclusionIn this study, regimens that contained at least 600,000 IU of ergocalciferol appeared to be the most effective in achieving vitamin D sufficiency. Guidelines for the treatment of vitamin D insufficiency in healthy adults should be developed. (Endocr Pract. 2009;15:95-103)     

Vitamin D supplements in pregnant Asian women: effects on calcium status and fetal growth.

In a double-blind trial of vitamin D supplements in pregnant Asian women calciferol (ergocalciferol, 1000 IU/day) was administered to 59 women and placebo to 67 controls during the last trimester. The two groups had similar distributions of maternal age, height, parity, number of vegetarians, countries of origin, and sex and gestation of the infants. At entry to the trial maternal serum 25-hydroxy vitamin D (25-OHD) concentrations were low in both treatment and control groups and significantly lower in vegetarians than non-vegetarians. Mothers in the treatment group gained weight faster in the last trimester than those in the control group, and at term they and their infants all had adequate plasma 25-OHD concentrations, Mothers and infants in the control group, however, had low plasma concentrations of 25-OHD and calcium and raised plasma alkaline phosphatase (bone isoenzyme) activity. Five of these infants developed symptomatic hypocalcaemia. Almost twice as many infants in the control group were small for gestational age (29% v 15%), but there were no significant differences between the two groups of infants in antropometric measurements. Infants in the control group, however, had larger fontanelles, suggesting impaired ossification of the skull. Because of the benefits to mothers and infants in the treatment group and the absence of side effects, vitamin D supplements should be given to all pregnant Asian women in the United Kingdom.     

Metabolism of vitamin D2 in pig liver homogenates: evidence for a free radical reaction

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.     

Effect of vitamin D3 administration on serum 25-hydroxyvitamin D3, 1,25-dihydroxyvitamin D3 and osteocalcin in vitamin D-deficient elderly people

In elderly institutionalized people, confined to bedroom and receiving no vitamin D supplementation, the frequency of vitamin D deficiency is found very high. Systematic administration of vitamin D has, therefore, been proposed to correct vitamin D deficiency. Within this context, we studied 40 elderly institutionalized subjects (mean age 80.5 + 7.2 yr) with low 25(OH)D3 concentrations (4.4 + 1.8 micrograms/l). Sixteen of them (Group I) had low serum calcium concentrations (less than 2.3 mmol/l) and 24 (Group II) had normal serum calcium concentrations (from 2.3 to 2.6 mmol/l). As hypocalcemia has been shown to regulate 1,25(OH)D3 production independent of PTH in animals and in humans, we compared their respective responses to the administration of vitamin D3. Subjects received a total dose of 15 mg (600,000 IU) of vitamin D3 divided into 3 i.m. injections at one month intervals and were explored before therapy and one and 6 months after the last dose of vitamin D3. The treatment induced a similar marked rise in 25(OH)D3 levels (from 4.1 + 1.7 to 24.4 + 8.7 micrograms/l for group I and from 5.1 + 1.8 to 27.2 + 8.0 micrograms/l for group II) in both groups but increased the 1,25(OH)2D3 concentrations only in group I (from 22.9 + 6.9 to 32.6 + 11.3 ng/l). Meanwhile serum calcium concentrations rose in group I (to low normal range i.e. 2.31 + 0.07 mmol/l) and were unaffected in group II. These results suggest that hypocalcemia is a potent stimulator of renal 1-hydroxylase in elderly people. Furthermore, a transient significant (P less than 0.01) increase in serum osteocalcin (from 10.6 + 4.1 to 14.1 + 5.9 micrograms/l) could be observed in group I which demonstrates for the first time that the osteocalcin response of osteoblasts to stimulation by 1,25(OH)2D3 is retained in very old people.     

Determination of the affinity of vitamin D metabolites to serum vitamin D binding protein using assay employing lipid-coated polystyrene beads.

We have developed an assay to measure the affinity of serum vitamin D binding protein for 25-hydroxyvitamin D3, 1,25-dihydroxyvitamin D3, and vitamin D3, using uniform diameter (6.4 microns) polystyrene beads coated with phosphatidylcholine and vitamin D metabolites as the vitamin D donor. The lipid metabolite coated beads have a solid core, and thus all of the vitamin D metabolites are on the bead surface from which transfer to protein occurs. After incubating these beads in neutral buffer for 3 h, essentially no 3H-labeled vitamin D metabolites desorb from this surface. Phosphatidylcholine/vitamin D metabolite-coated beads (1 microM vitamin D metabolite) were incubated with varying concentrations of serum vitamin D binding protein under conditions in which the bead surfaces were saturated with protein, but most of the protein was free in solution. After incubation, beads were rapidly centrifuged without disturbing the equilibrium of binding and vitamin D metabolite bound to sDBP in solution was assayed in the supernatant. All three vitamin D metabolites became bound to serum vitamin D binding protein, and after 10 min of incubation the transfer of the metabolites to serum vitamin D binding protein was time independent. The transfer followed a Langmuir isotherm, and the Kd for each metabolite binding to serum vitamin D binding protein was derived by nonlinear least-squares fit analysis. From this analysis the following values for the Kd were obtained: 5.59 x 10(-6) M, 25-hydroxyvitamin D; 9.45 x 10(-6) M, 1,25-dihydroxyvitamin D; and 9.17 x 10(-5) M, vitamin D. In conclusion, we have developed a method which avoids problems encountered in previous assays and allows the precise and convenient determination of binding affinities of vitamin D metabolites and serum vitamin D binding protein.     

The vitamin D system in iguanian lizards

The apparent plasma concentration of vitamin D binding protein (DBP) in an iguanian lizard, Pogona barbata, and the affinity of this protein for 25-hydroxyvitamin D₃ (25(OH)D₃), 25-hydroxyvitamin D₂ (25(OH)D₂), and 1,25-dihydroxyvitamin D₃ (1,25(OH)D₃) was found to resemble more closely that of the domestic hen than that of the human. The characteristics of Pogona DBP, the pattern of vitamin D metabolites derived from injected radioactive vitamin D₃ and the plasma concentrations of endogenous 25-hydroxyvitamin D (25(OH)D) in a range of iguanian lizards have been examined. The findings suggest that 25-hydroxyvitamin D (25(OH)D) is the major metabolite of vitamin D, and that it may represent the storage form of vitamin D in these species in the same way as in mammals. High concentrations of vitamin D within iguanian embryos and egg yolks suggest a role for this compound in embryogenesis in these species, and perhaps indicates that there is a mechanism for vitamin D delivery to eggs comparable to that found in the domestic chicken.     

Effects of phytase and 25-hydroxyvitamin D3 inclusions on the performance, mineral balance and bone parameters of grower-finisher pigs fed low-phosphorus diets

Two experiments, a performance experiment and a mineral balance study, were conducted on grower-finisher pigs (42 to 101 kg live weight) to investigate the effects of Peniophora lycii phytase enzyme and 25-hydroxyvitamin D3 (25-OHD3) on growth performance, carcass characteristics, nutrient retention and excretion, and bone and blood parameters. The two experiments were designed as a 2 × 2 factorial (two levels of phytase and two levels of 25-OHD3). The four diets were T1, low-phosphorous diet; T2, T1 + phytase; T3, T1 + 25-OHD3 and T4, T1 + phytase + 25-OHD3 diet. In all, 25 μg of 25-OHD3 was used to replace 1000 IU of vitamin D3 in diets T3 and T4. Diets were pelleted (70°C) and formulated to contain similar concentrations of energy (13.8 MJ DE/kg), lysine (9.5 g/kg) and digestible phosphorus (P; 1.8 g/kg). Neither the inclusion of phytase nor 25-OHD3 in the diet had any effect on pig performance. There was an interaction between phytase and 25-OHD3 on calcium (Ca) and P retention (P < 0.01) and on the apparent digestibility of ash (P < 0.01), P (P < 0.001) and Ca (P < 0.001). Pigs offered phytase diets only, had a higher retention of Ca and P and digestibility of ash (P < 0.01), P (P < 0.001) and Ca (P < 0.01) compared with pigs offered unsupplemented diets. However, when the combination of phytase and 25-OHD3 were offered, no effects were detected compared with 25-OHD3 diets only. Pigs fed phytase diets had higher bone ash (P < 0.01), bone P (P < 0.01) and bone Ca (P < 0.05) concentrations compared with pigs offered non-phytase diets. In conclusion, pigs offered phytase diets had a significantly increased bone ash, Ca and P than pigs offered unsupplemented phytase diets. However, there was no advantage to offering a combination of phytase and 25-OHD3 on either bone strength or mineral status compared to offering these feed additives separately.