Determination of vitamin D3 conjugated metabolites: a complementary view on hydroxylated metabolites.

Experts typically define vitamin D deficiency levels by the determination of a circulating 25-hydroxyvitamin D3-calcifediol prohormone. A large part of the population is characterized by deficient vitamin D levels (calcifediol < 20 ng mL-1) despite individuals not being affected by any disorder. Cholecalciferol (vitamin D3) and/or calcifediol supplementation is a common practice for vitamin D-deficient individuals as recommended by international scientific societies and official agencies. In the last few years, several studies have reported the presence of conjugated vitamin D3 metabolites, mainly glucuronidation and sulfation derivatives, although simultaneous quantitative measurements involving phase I and II vitamin D metabolites have not been carried out. A quantitative method based on tandem mass spectrometry detection is proposed here for the combined determination of phase I and phase II vitamin D3 metabolites in human serum. As phase I and phase II metabolites are preferentially ionized in different modes, a switching polarity mode was adopted to determine both groups of compounds in serum at high sensitivity levels (pg mL-1). The validation of this proposal was successfully accomplished by following the Center for Drug Evaluation and Research (CDER) guidelines. Its applicability was tested in a cohort of volunteers with mostly deficient baseline levels. Considering the sulfated form of calcifediol, the sum of its concentrations showed sufficient baseline vitamin D levels in all individuals, suggesting that this could be a novel strategy for vitamin D deficiency definition. Therefore, phase II metabolites are proposed to be included when evaluating the vitamin D status since they provide more information about the overall status of the vitamin D endocrine system. Nevertheless, further studies are required to confirm the biological activity of these conjugated metabolites and the suitability of this strategy for the description of vitamin D deficiency.

Validation and Determination of 25(OH) Vitamin D and 3-Epi25(OH)D3 in Breastmilk and Maternal- and Infant Plasma during Breastfeeding.

Vitamin D deficiency in pregnant women and their offspring may result in unfavorable health outcomes for both mother and infant. A 25hydroxyvitamin D (25(OH)D) level of at least 75 nmol/L is recommended by the Endocrine Society. Validated, automated sample preparation and liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods were used to determine the vitamin D metabolites status in mother-infant pairs. Detection of 3-Epi25(OH)D3 prevented overestimation of 25(OH)D3 and misclassification of vitamin D status. Sixty-three percent of maternal 25(OH)D plasma levels were less than the recommended level of 25(OH)D at 3 months. Additionally, breastmilk levels of 25(OH)D decreased from 60.1 nmol/L to 50.0 nmol/L between six weeks and three months (p < 0.01). Furthermore, there was a positive correlation between mother and infant plasma levels (p < 0.01, r = 0.56) at 3 months. Accordingly, 31% of the infants were categorized as vitamin D deficient (25(OH)D < 50 nmol/L) compared to 25% if 3-Epi25(OH)D3 was not distinguished from 25(OH)D3. This study highlights the importance of accurate quantification of 25(OH)D. Monitoring vitamin D metabolites in infant, maternal plasma, and breastmilk may be needed to ensure adequate levels in both mother and infant in the first 6 months of infant life.

Correlations between Vascular Stiffness Indicators, OPG, and 25-OH Vitamin D3 Status in Heart Failure Patients.

Background and objectives: The purpose of the study is to correlate vascular calcification biomarkers osteoprotegerin (OPG) and 25-hydroxyvitamin D3 (25-OH-D3), indicators of arterial stiffness carotid-femoral pulse wave velocity (c-f PWV) and renal resistive index (RRI), with parameters of left ventricular function in heart failure patients versus control. Materials and methods: Our case-control study compared 60 patients with ischemic heart failure and reduced left ventricular ejection fraction (LVEF) (<40%) with a control group of 60 healthy age-matched subjects (CON). Serum levels of OPG and 25-OH-D3 were determined by ELISA. Left ventricular volumes (LVESV, LVEDV) and LVEF were measured by echocardiography. C-f PWV was determined using the arteriograph device. RRI was measured by duplex Doppler. Peak systolic velocity (PSV) and minimum end-diastolic velocity (EDV) were determined using angle correction. The estimated glomerular filtration rate (eGFR) was calculated using the MDRD equation. The Pearson's correlation coefficient was used for interpretation of results. Results: OPG values were significantly higher in heart failure (HF) patients vs. CON (4.7 ± 0.25 vs. 1.3 ± 0.67 ng/mL, p < 0.001). 25-OH vitamin D3 levels were significantly lower in HF patients vs. CON (20.49 ± 7.31 vs. 37.09 ± 4.59 ng/mL, p < 0.001). Multiple regression analysis considering 25-OH D3 as a dependent variable demonstrated indicators of vascular stiffness RRI, c-f PWV and vascular calcification biomarker OPG as predictors. OPG values were significantly correlated with cardiac parameters LVEDV (r = 0.862, p < 0.001), LVEF (r = -0.832, p < 0.001), and c-f PWV(r = 0.833, p < 0.001), and also with 25-OH-D3 (r = -0.636, p < 0.001). RRI values were significantly correlated with cardiac parameters LVEDV (r = 0.586, p < 0.001) and LVEF (r = -0.587, p < 0.001), and with eGFR (r = -0.488, p < 0.001), c-f PWV(r = 0.640, p < 0.001), and 25-OH-D3 (r = -0.732, p < 0.001). Conclusions: This study showed significant correlations between vitamin D deficit and vascular stiffness indicators in heart failure patients with reduced ejection fraction, demonstrating the importance of these examinations for a better evaluation of these patients. Together with the evaluation of renal function, the measurement of vascular stiffness indicators and biomarkers might play a key role in identifying patients at greater risk for worsening disease prognosis and for shorter life expectancy, who could benefit from vitamin D supplementation. The abstract was accepted for presentation at the Congress of the European Society of Cardiology, Munich, 2018.

The use of synthetic and natural vitamin D sources in pig diets to improve meat quality and vitamin D content.

This study investigated the effects of synthetic and natural sources of vitamin D biofortification in pig diets on pork vitamin D activity and pork quality. One hundred and twenty pigs (60 male, 60 female) were assigned to one of four dietary treatments for a 55 d feeding period. The dietary treatments were (1)50 µg vitamin D3/kg of feed; (2)50 µg of 25-hydroxvitamin D3/kg of feed (25-OH-D3); (3)50 µg vitamin D2/kg of feed; (4)50 µg vitamin D2-enriched mushrooms/kg of feed (Mushroom D2). The pigs offered the 25-OH-D3 diet exhibited the highest (P < 0.001) serum total 25-hydroxyvitamin D concentration and subsequently exhibited the highest (P < 0.05) Longissimus thoracis (LT) total vitamin D activity. Mushroom D2 and 25-OH-D3 supplementation increased pork antioxidant status. The vitamin D2-enriched mushrooms improved (P < 0.05) pig performance, carcass weight and LT colour. In conclusion, 25-OH-D3 is the most successful source for increasing pork vitamin D activity, while Mushroom D2 may be a new avenue to improve animal performance and pork quality.

Vitamin D-biofortified beef: A comparison of cholecalciferol with synthetic versus UVB-mushroom-derived ergosterol as feed source.

This study investigates dietary fortification of heifer feeds with cholecalciferol and ergocalciferol sources and effects on beef total vitamin D activity, vitamer, respective 25-hydroxymetabolite contents, and meat quality. Thirty heifers were allocated to one of three dietary treatments [(1) basal diet + 4000 IU of vitamin D3 (Vit D3); (2) basal diet + 4000 IU of vitamin D2 (Vit D2); and (3) basal diet + 4000 IU of vitamin D2-enriched mushrooms (Mushroom D2)] for a 30 day pre-slaughter period. Supplementation of heifer diets with Vit D3 yielded higher (p < 0.001) Longissimus thoracis (LT) total vitamin D activity (by 38-56%; p < 0.05) and serum 25-OH-D concentration (by 20-36%; p < 0.05), compared to that from Vit D2 and Mushroom D2 supplemented animals. Irrespective of vitamin D source, carcass characteristics, sensory and meat quality parameter were unaffected (p > 0.05) by the dietary treatments. In conclusion, vitamin D3 biofortification of cattle diets is the most efficacious way to enhance total beef vitamin D activity.

Effect of dietary vitamin D3 and 25-hydroxyvitamin D3 supplementation on plasma and milk 25-hydroxyvitamin D3 concentration in dairy cows.

Milk enriched with vitamin D by supplementing dairy cow diets could provide a valuable dietary source of vitamin D, but information on the feasibility of this approach is limited. In the current study, the effects of supplementing dairy cows with either vitamin D3 or 25(OH)D3 over the transition/early lactation period on plasma and milk vitamin D concentrations were compared. Sixty dairy cows were randomly allocated to 1 of 4 dietary treatments from 14 d precalving to 56 d postcalving. Treatments were a control diet (control) for both precalving and postcalving periods containing 0.625 mg/d of vitamin D3; a precalving diet supplemented with 6 mg of 25(OH)D3/d, but with a postcalving diet matching that of the control diet [25(OH)D3 precalving]; the control diet precalving but with the postcalving diet supplemented with 2 mg of vitamin D3/d (D3max), and the control diet precalving but with the postcalving diet supplemented with 1.5 mg of 25(OH)D3/d [25(OH)D3 postcalving]. No treatment effect on milk yield, composition or 25(OH)D3 concentration was observed. However, an interaction was observed of treatment and time for plasma 25(OH)D3 concentration; this increased within 2 wk of supplementation for the 25(OH)D3 precalving treatment (peaking just after calving, 202 ng/mL), whereas that of the 25(OH)D3 postcalving group had a slower response following supplementation, continuing to increase at 56 d. Correlations were observed between plasma and milk 25(OH)D3 concentrations at d 4 and 14 of lactation, but not at later sampling times. The D3max treatment did not increase 25(OH)D3 concentration in plasma or milk. Overall, results from this study indicate that supplemental 25(OH)D3 is an effective means of enhancing dairy cow plasma 25(OH)D3 concentrations compared with vitamin D3 supplementation, but not necessarily milk concentrations.

Highly sensitive detection of 25-HydroxyvitaminD3 by using a target-induced displacement of aptamer.

For the prevention of 25-HydroxyvitaminD3 deficiency, in this study, aptamers which can bind to 25-HydroxyvitaminD3 with high specificity and affinity, were successfully developed by using immobilization-free, graphene oxide-based systemic evolution of ligands by exponential enrichment (GO-SELEX) method. The 9 sequences including VDBA14 aptamer were obtained out of 16 aptamer candidates, based on the specificity and affinity of the aptamers confirmed by both the gold nanoparticles (AuNPs)-based colorimetric assay and the isothermal titration calorimetry (ITC) method. Among them, the aptamer, VDBA14, developed in this study was found to show a great affinity to 25-HydroxyvitaminD3, with 11nM of its Kd value. Moreover, the circular dichroism (CD) analysis data indicated the target-induced displacement of the aptamer VDBA14clearly. In addition, this target-induced change of the aptamer was also confirmed again by conducting two different experimental formats, the use of streptavidin-coated 96-well plates and the use of magnetic beads. The results clearly indicated that the structure of VDBA14 aptamer was changed upon the binding of the target, 25-HydroxyvitaminD3, and so the indicator sequences (partially complementary to the aptamer sequence) tagged with an enzyme as a signaling molecule could be de-hybridized from the aptamer. Finally, the limit of detection for vitamin D based on AuNPs-based colorimetric assay using VDBA14 aptamer was found to be 1µM. All these results were taken together, the aptamer which was developed could play an exquisite role in the fields of early medical diagnosis of vitamin D deficiency with accurate, rapid and simple analytical method.

[A Case of Tenofovir-associated Fanconi Syndrome in Patient with Chronic Hepatitis B].

Tenofovir disoproxil fumarate (TDF) is one of the most widely used treatment options for human immunodeficiency virus (HIV) and HBV infections. Despite its efficacy and safety, some cases of nephrotoxicity have been reported in the treatment of HIV patients. Even more recently, very few cases of Fanconi syndrome associated with tenofovir therapy in HBV monoinfection have been reported. Herein, we report a case of a 47-year-old male with an HBV monoinfection, who developed Fanconi syndrome and a secondary osteomalacia with multiple bone pain. After TDF withdrawal and supplementation of calcitriol, his renal function was reverted. Although the overall risk of TDF-associated nephrotoxicity is very low, both glomerular and tubular function should be monitored in patients undergoing TDF treatment.

Oral intake of 7-dehydrocholesterol increases vitamin D3 concentrations in the liver and kidney.

INTRODUCTION: Due to the high prevalence of vitamin D deficiency, strategies are needed to improve vitamin D status. Food components can affect vitamin D metabolism and have to be considered when estimating the efficacy of vitamin D supplements. 7-dehydrocholesterol (7-DHC) occurs naturally in food, but its impact on vitamin D metabolism has not yet been examined. METHODS: Three groups of male C57BL/6 mice (n=12 per group) were placed on a diet that contained 0, 2.5 or 5mg 7-DHC per kg diet over a period of 6 weeks. Vitamin D and other sterols in the serum, skin, liver and kidney were quantified by LC-MS/MS. The relative mRNA abundance of hepatic genes encoding vitamin D hydroxylation enzymes and transporters was analyzed by real-time RT-PCR. RESULTS: We found a substantial dose-dependent increase of non-hydroxylated vitamin D3 in the liver and kidney of mice fed a diet containing 7-DHC. The vitamin D3 content in the liver was 2.80±0.61pmol/g, 7.34±4.28pmol/g and 12.9±3.58pmol/g in groups that received 0, 2.5 and 5mg/kg 7-DHC, respectively. In the kidney, the vitamin D3 content of these groups was 1.78±1.17pmol/g, 3.55±1.06 and 6.36±2.29pmol/g, respectively. The serum and tissue concentrations of 25-hydroxyvitamin D3 (25(OH)D3) remained unaffected by 7-DHC. The relative mRNA data provided no plausible mechanism for the observed effects of 7-DHC on vitamin D3. All groups of mice had similar concentrations of cholesterol, desmosterol and 7-DHC in their serum and tissues. CONCLUSION: The current findings provide the first evidence that dietary 7-DHC seems to affect vitamin D metabolism. The underlying mechanism remains elusive and needs further investigation.

Effect of feeding 25-hydroxyvitamin D3 with a negative cation-anion difference diet on calcium and vitamin D status of periparturient cows and their calves.

Holstein cows (>1 gestation) were fed 1 of 3 diets during the last 13 d of gestation (ranged from 22 to 7 d). The control diet (16 cows) was formulated to provide 18,000 IU/d of vitamin D3 and had a dietary cation-anion difference (DCAD) of 165mEq/kg (DCAD=Na + K - Cl - S). The second diet (DCAD + D) provided the same amount of vitamin D3 but had a DCAD of -139mEq/kg (17 cows). The third diet (DCAD + 25D) had no supplemental vitamin D3 but provided 6mg/d of 25-(OH) vitamin D3 [25-(OH)D3] with a DCAD of -138mEq/kg (20 cows). Diets were fed until parturition and then all cows were fed a common lactation diet that contained vitamin D3. Negative DCAD diets reduced urine pH, with the greatest decrease occurring with the DCAD + D treatment. Urinary Ca excretion was greatest for cows fed DCAD + 25D followed by cows fed DCAD + D. Urinary pH was negatively correlated with urinary excretion of Ca for cows fed DCAD + D. No such correlation was observed with the DCAD + 25D treatment because substantial excretion of urinary Ca occurred at moderate urinary pH values for that treatment. Cows fed DCAD + 25D had greater serum concentrations of 25-(OH)D3 than other treatments from 5 d after supplementation started through 7 d in milk. Concentrations of 1,25-(OH)2D3 in serum were greatest in DCAD + 25D cows starting at 2 d before calving and continued through 7 d in milk. Serum Ca concentrations 5 d before calving were greatest for cows fed DCAD + 25D, but at other time points before and after parturition treatment did not affect serum Ca. Incidence of clinical hypocalcemia was not statistically different between treatments, but cows fed DCAD + 25 had the highest incidence rate (12.5, 0, and 20% for control, DCAD + D, and DCAD + 25D). Calves born from cows fed DCAD + 25D had greater concentrations of 25-(OH)D3 in serum at birth than calves from other treatments (before colostrum consumption), but concentrations were similar by 3 d of age. Concentrations of 25-(OH)D3 in colostrum and transition milk were increased by feeding DCAD + 25D, but by 28 d in milk treatment effects no longer existed. Overall, feeding 25-OH vitamin D with a negative DCAD diet increased vitamin D status of the cow and her newborn calf but had minimal effects on calcium status and did not have positive effects on the incidence of hypocalcemia.

Analytical measurement and clinical relevance of vitamin D(3) C3-epimer.

With an ever-increasing clinical interest in vitamin D insufficiency, numerous automated immunoassays, protein binding assays, and in-house LC-MS/MS methods are being developed for the quantification of 25-hydroxyvitamin D(3) (25(OH)D(3)). Recently, LC-MS/MS methods have identified an epimeric form of 25(OH)D(3) that has been shown to contribute significantly to 25(OH)D(3) concentration, particularly in infant populations. This review describes the metabolic pathway and physiological functions of 3-epi-vitamin D, compares the capability of various 25(OH)D(3) methods to detect the epimer, and highlights recent publications quantifying 3-epi-25(OH)D(3) in infant, pediatric, and adult populations. In total, this review summarizes the information necessary for clinicians and laboratorians to decide whether or not to report/consider the C3-epimer in the analysis and clinical assessment of vitamin D status.

Serum levels of 3-epi-25-OH-D3 during hypervitaminosis D in clinical practice.

CONTEXT: Intoxication from vitamin D supplements has been rarely reported, but nowadays, it occurs more frequently. The presence of the C-3 epimer of 25-hydroxyvitamin D(3) (3-epi-25-OH-D(3)) is highly prevalent in adults, although there is little information regarding its in vivo relevance, if any, especially under pathological conditions. OBJECTIVE: Our aim was to assess the presence of the 3-epi-25-OH-D(3) in serum samples displaying 25-OH-D(3) concentrations indicative of hypervitaminosis D. DESIGN, SETTING, PATIENTS, AND MAIN OUTCOME MEASURE: A total of 58 samples displaying a wide range of concentrations of 25-OH-D(3) (>64-439 ng/ml) by ultrafast liquid chromatography were consecutively recruited and reassessed for the presence of 3-epi-25-OH-D(3) using a second chromatographic system. Data from additional biochemical tests performed as part of the patient evaluation were also recorded. RESULTS: Mean relative contribution of 3-epi-25-OH-D(3) was less than 4%, and concentrations ranged from 2-28.6 ng/ml. Serum levels of the C3 epimer, but not the relative contribution, correlate with serum 25-OH-D(3). Overall, in subjects with 25-OH-D(3) concentrations indicative of hypervitaminosis D, the presence of the C-3 epimer and its levels were apparently unrelated to age, serum markers of renal and liver function, acute-phase reactants, and the presence of hypercalcemia. 3-Epi-25-OH-D(3) did not correlate with PTH, but subjects displaying PTH suppression (<14 pg/ml) showed higher concentrations of 3-epi-25-OH-D(3). CONCLUSION: The relative contribution of 3-epi-25-D(3) was not significantly altered during hypervitaminosis D, although the absolute levels reached in serum may be biologically relevant. From a clinical viewpoint, although the small size of the group may affect the lack of relationships, the presence of 3-epi-25-OH-D(3) was apparently unrelated to serum markers of renal and liver function, acute-phase reactants, PTH, and the presence of hypercalcemia.

Rapid determination of 25-hydroxy vitamin D3 in swine tissue using an isotope dilution HPLC-MS assay.

A rapid method for quantification of 25-hydroxy vitamin D3 in different swine tissues based on isotope dilution HPLC-MS has been developed and validated. Six times deuterated analyte is used as internal standard. The method is fast and can be performed with only 1g sample. Sample preparation for kidney, liver, muscle and spleen requires only homogenisation and extraction with methanol. An additional enzymatic digest is required for skin, and clean-up of the extract by solid-phase extraction (SPE) is used for adipose tissue and skin. The lower limit of detection varies from 1 ng/g (muscle) to 5 ng/g (adipose and skin). The method has been successfully applied to various tissue samples of pigs fed for 119 days either 2000 IU of vitamin D3 or 50 microg of 25-hydroxy vitamin D3 per kg feed. For animals ingesting 25-OH-D3 supplements the highest tissue contents were observed in the skin (24.8+/-3.5 ng/g), followed by kidney (14.2+/-1.5 ng/g), liver and muscle (5.7+/-0.6 ng/g). The 25-OH-D3 content in the skin was significantly higher in animals ingesting 2000 IU/kg of vitamin D3 (39.5+/-13.4 ng/g). Levels in selected tissues of some animals were below the lower limit of quantification. No measurable amounts of 25-OH-D3 were found in spleen, abdominal fat and subcutaneous fat of the animals of both groups as well as in the liver, kidney and muscle of the animals ingesting 2000 IU/kg of vitamin D3.

Liquid chromatography-tandem mass spectrometric method for the determination of salivary 25-hydroxyvitamin D3: a noninvasive tool for the assessment of vitamin D status.

A sensitive liquid chromatography-electrospray ionization-tandem mass spectrometric (LC-ESI-MS/MS) method for the determination of 25-hydroxyvitamin D(3) [25(OH)D(3)] in human saliva has been developed and validated. The saliva was deproteinized with acetonitrile, purified using a Strata-X cartridge, derivatized with a Cookson-type reagent, 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD), and subjected to LC-MS/MS. The PTAD derivative was much more easily ionized in positive-ESI-MS and efficiently produced a characteristic product ion during MS/MS, compared to the intact 25(OH)D(3). Methylamine was used as the mobile phase additive, and also effectively enhanced the assay sensitivity. Quantification was based on selected reaction monitoring, and 25-hydroxyvitamin D(4) was used as the internal standard. This method allowed the reproducible and accurate quantification of salivary 25(OH)D(3) using a 1.0-ml sample, and the limit of quantitation for 25(OH)D(3) was 2.0 pg/ml. The applicability of the developed method for clinical studies was then examined. There was a positive linear relationship (r (2) = 0.830) between the serum 25(OH)D(3) level, which is conventionally used as a means of assessing the vitamin D status, and the salivary 25(OH)D(3) level measured using the proposed method. The method also enabled the detection of the increase in the salivary 25(OH)D(3) level after the supplementation of vitamin D(3).

Management of chronic kidney disease mineral-bone disorder.

Chronic kidney disease mineral-bone disorder (CKD-MBD) is a systemic disorder of abnormal serum levels of mineral-related biochemistries, abnormal bone, and extraskeletal calcification. Although we have gained understanding on how these components are interrelated, our therapeutic tools remain focused on only one aspect of CKD-MBD at a time. However, the management of these disorders is also interrelated; treatments may help one aspect of the disorder but cause or accelerate another. As such, management remains a major challenge to nephrologists and requires balancing risk and benefit of the various available therapies. Our challenge for the decade ahead is to determine which combinations of therapy can be used safely together to prevent morbidity and mortality in CKD. Furthermore, the pathophysiology that sets these events into motion begins well before the onset of ESRD. Future therapies and guidelines should, therefore, also emphasize the need for earlier detection and management of CKD, shaped by the results of valid clinical trials.

Supplemental vitamin D3 concentration and biological type of steers. II. Tenderness, quality, and residues of beef.

Vitamin D3 was orally supplemented to determine the supplemental dose that improved beef tenderness in different cattle breed types. Feedlot steers (n = 142) were arranged in a 4 x 3 factorial arrangement consisting of four levels of supplemental vitamin D3 (0, 0.5, 1, and 5 million IU/steer daily) administered for eight consecutive days antemortem using three biological types (Bos indicus, Bos Taurus-Continental, and Bos Taurus-English). Warner-Bratzler shear force (WBSF) was measured at 3, 7, 10, 14, and 21 d postmortem, and trained sensory analysis was conducted at 7 d postmortem on LM, semimembranosus, gluteus medius, and supraspinatus steaks. Concentrations of vitamin D3 and the metabolites 25-hydroxyvitamin D3, and 1,25-dihydroxyvitamin D3 were determined in the LM, liver, kidney, and plasma. Biological type of cattle did not interact (P > 0.10) with vitamin D3 supplementation for sensory or tenderness traits, suggesting that feeding vitamin D3 for 8 d before slaughter affected the different biological types of cattle similarly. Supplementing steers with 0.5, 1, or 5 million IU/(steer(d) decreased (P < 0.05) LM WBSF at 7, 10, 14, and 21 d postmortem compared with controls, and vitamin D3 treatments of 0.5, 1, and 5 million IU decreased (P < 0.05) semimembranosus WBSF at 3, 7, and 14 d postmortem. In general, vitamin D3-induced improvements in WBSF were most consistent and intense in LM steaks. Sensory panel tenderness was improved (P < 0.05) by all vitamin D3 treatments in LM steaks. Sensory traits ofjuiciness, flavor, connective tissue, and off-flavor were not (P > 0.05) affected by vitamin D3 treatments. All vitamin D3 treatments decreased micro-calpain activity and increased muscle Ca concentrations (P < 0.05). Vitamin D3 concentrations were increased (P < 0.05) by supplementation in all tissues tested (liver, kidney, LM, and plasma); however, cooking steaks to 71 degrees C decreased (P < 0.05) treatment residue effects. The vitamin D metabolite 1,25-dihydroxyvitamin D3 was increased (P < 0.05) only in plasma samples as a result of the vitamin D3 treatments. These results indicate that supplementation with vitamin D3 at 0.5 million IU/steer daily for eight consecutive days before slaughter improved tenderness in steaks from different subprimal cuts by affecting muscle Ca concentrations, micro-calpain activities, and muscle proteolysis, with only a small effect on tissue residues of vitamin D3.

Cholecalciferol and 25-hydroxycholecalciferol content of chicken egg yolk as affected by the cholecalciferol content of feed.

The predominant source of vitamin D is the synthesis of cholecalciferol in the skin by the action of sunlight; however, due to the relative lack of sunlight, the intake of vitamin D from food is emphasized during winter, especially in the northern countries. Only a few foods (fish, eggs, wild mushrooms, meat, and milk) are natural sources of vitamin D. In addition, the content of vitamin D in foods is generally low, and some groups of people obtain amounts of vitamin D that are too small from their diet. The present study was designed to determine whether it is possible to increase the vitamin D content of egg yolk by giving hens feed containing elevated levels of cholecalciferol. Three cholecalciferol levels were tested: 26.6 (1064), 62.4 (2496), and 216 microgram (8640 IU)/kg feed. Egg yolk samples were taken after 0, 4, 5, and 6 weeks and were assayed for the presence of cholecalciferol and 25-hydroxycholecalciferol using an HPLC method. According to the present study, there was strong positive correlation between cholecalciferol content in poultry feed and cholecalciferol (r = 0. 995) and 25-hydroxycholecalciferol (r = 0.941) content in egg yolk.

Vitamin D levels in noninstitutionalized children with cerebral palsy.

Calcitriol (1,25-dihydroxy vitamin D) is an important hormone in calcium and phosphate metabolism. Levels of calcitriol and its precursor, 25-hydroxy vitamin D (calcidiol), were measured in a heterogeneous group of 125 noninstitutionalized children and adolescents with spastic cerebral palsy. Levels of each were correlated with: (1) clinical factors including mobility, prior fracture, and use of anticonvulsants; (2) nutrition and growth parameters including skinfolds, body mass index, and use of vitamin supplements; and (3) other serum analyses including osteocalcin as a marker of bone formation, calcium, and alkaline phosphatase. Levels of calcidiol and calcitriol did not correlate with any of the various clinical, nutritional, or growth parameters examined. The prevalence of low (< 10 ng/mL) levels of calcidiol was significant (19%), and dependent on the season of the year in which the level was measured. In contrast, less than 2% of the patients were found to have a low (< 20 pg/mL) level of calcitriol and the mean was comparable to normal pediatric subjects. Levels of calcitriol are maintained in noninstitutionalized children with cerebral palsy despite anticonvulsants, poor nutrition, and calcidiol levels that vary greatly with the seasons.

Vitamin D3 and its metabolites in the tomato plant.

The tomato plant has been demonstrated to have vitamin D-like activity. The activity was present in the leaves but not in the fruit of the plant. The chloroform extract of the leaves (containing free vitamin D and its metabolites) and the ethanol extract of the residue (containing the glycosidic forms) were partially purified by column chromatography. The fractions corresponding to authentic vitamin D3, 25-hydroxy vitamin D3 and 1,25-dihydroxy vitamin D3 were tested for biological activity and analysed by HPLC. The results indicate that the plant contains vitamin D3, 25-hydroxy vitamin D3 and 1,25-dihydroxy vitamin D3 and their glycosidic forms. Free vitamin D3 was observed to be the major active principle and the concentration of the free forms of the metabolites was higher than the corresponding glycosides.

25-Hydroxyvitamin D and vitamin D in human milk: effects of supplementation and season.

Breast-milk 25-hydroxyvitamin D (25-[OH]D) and vitamin D were measured in mothers supplemented with 2000 or 1000 IU (50 or 25 micrograms) of vitamin D/d or with no supplementation. Fore- and hindmilk samples were collected at two stages of lactation (8 and 15 or 20 wk after delivery) and at different seasons. Season affected the levels of 25-(OH)D and vitamin D. The 25-(OH)D levels were higher in hind- than in foremilk. Supplementation had no effect on vitamin D levels. Milk 25-(OH)D levels of mothers receiving either 1000 or 2000 IU (25 or 50 micrograms) vitamin D/d were significantly higher than those of unsupplemented mothers in February and April. In theory, supplementation with 2000 IU (50 micrograms) vitamin D should have increased the calculated antirachitic activity of the milk in winter to the levels of unsupplemented mothers in September; however, responses varied widely among individuals.