Vitamin D composition of Australian foods.

Australia needs accurate vitamin D food composition data to support public health initiatives. Previously, limitations in analytical methodology have precluded development of a comprehensive database. We used liquid chromatography with triple quadrupole mass spectrometry (LC-QQQ) to analyse 149 composite samples representing 98 foods (primary samples n = 896) in duplicate for vitamin D3, 25-hydroxyvitamin D3 (25(OH)D3), vitamin D2, 25(OH)D2. The greatest concentrations of vitamin D3 were found in canned salmon and a malted chocolate drink powder (fortified); chicken eggs and chicken leg meat contained the most 25(OH)D3. Margarine (fortified) and chocolate contained the greatest concentrations of vitamin D2, with smaller amounts found in various meat products. 25(OH)D2 was detected in various foods, including meats, and was quantitated in lamb liver. These data advance knowledge of dietary vitamin D in Australia and highlight the importance of analysis of these four forms of vitamin D to accurately represent the vitamin D content of food.

Consolidation of vitamin A and E methods onto a multiplexing liquid chromatography tandem mass spectrometry platform simplifies laboratory workflow.

BACKGROUND: Vitamin A and E are routinely monitored to assess nutritional status. The most commonly used approach for their measurement involves laborious liquid-liquid extraction followed by high-performance liquid chromatography (HPLC) analysis on dedicated instrumentation. We describe a simple, rapid protocol for measurement of vitamin A and E and their integration into an existing online sample preparation liquid chromatography tandem mass spectrometry (SPLC-MS/MS) workflow. METHODS: We performed a method comparison between the SPLC-MS/MS and HPLC methods for vitamin A and E by measuring patient specimens across the concentration range 11-81 µg/dL for vitamin A and 1-18 mg/L for vitamin E. The analysis times on each platform were also compared. RESULTS: SPLC-MS/MS and HPLC methods were comparable with regards to analytical performance; mean bias across the measured range was 2.54% (95% CL: -11.56-16.64%) for vitamin A and -2.04% (95% CL: -18.20-14.12%) for vitamin E. Total analysis times were 7 min and 15 min for SPLC-MS/MS and HPLC respectively. CONCLUSIONS: The development of a simplified sample preparation protocol and the use of multiplexing SPLC-MS/MS have reduced sample analysis times for vitamin A and E. This method has also optimized clinical workflow through consolidation of previously independent benches.

A novel caged Cookson-type reagent toward a practical vitamin D derivatization method for mass spectrometric analyses.

RATIONALE: 25-Hydroxylated vitamin D is the best marker for vitamin D (VD). Due to its low ionization efficiency, a Cookson-type reagent, 1,2,4-triazoline-3,5-dione (TAD), is used to improve the detection/quantification of VD metabolites by liquid chromatography/tandem mass spectrometry (LC/MS/MS). However, the high reactivity of TAD makes its solution stability low and inconvenient for practical use. We here describe the development of a novel caged Cookson-type reagent, and we assess its performances in the quantitative and differential detection of four VD metabolites in serum using LC/MS/MS. METHODS: Caged 4-(4'-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD) analogues were prepared from 4-(4'-dimethylaminophenyl)-1,2,4-triazolidine-3,5-dione. Their stability and reactivity were examined. The optimized caged DAPTAD (14-(4-(dimethylamino)phenyl)-9-phenyl-9,10-dihydro-9,10-[1,2]epitriazoloanthracene-13,15-dione, DAP-PA) was used for LC/MS/MS analyses of VD metabolites. RESULTS: The solution stability of DAP-PA in ethyl acetate dramatically improved compared with that of the non-caged one. We measured the thermal retro-Diels-Alder reaction enabling the release of DAPTAD and found that the derivatization reaction was temperature-dependent. We also determined the detection limit and the lower limit of quantifications for four VD metabolites with DAPTAD derivatization. CONCLUSIONS: DAP-PA was stable enough for mid- to long-term storage in solution. This advantage shall contribute to the detection and quantification of VD in clinical laboratories, and as such to the broader use of clinical mass spectrometry.

Half the Genetic Variance in Vitamin D Concentration is Shared with Skin Colour and Sun Exposure Genes.

This study assessed the heritability of 25 hydroxyvitamin D3 (25(OH)D3) in a large twin cohort and the shared effect of sun exposure and skin colour on 25(OH)D3 variance. Study participants included 1604 twin pairs and their siblings (n = 4020). Twin correlations for 25(OH)D3 concentration were rMZ=0.79 (584 pairs) and rDZ = 0.52 (1020 pairs) consistent with an average h2 = 0.50 throughout the year. Significant phenotypic and genetic seasonal fluctuation was observed in 25(OH)D3 concentrations with heritability decreasing during the winter (h2 = 0.37) compared to summer (h2 = 0.62). Skin colour (measured both ordinally and quantitatively) and self-reported sun exposure were found to significantly affect 25(OH)D3 concentration. Twins with olive/dark skin had significantly lower 25(OH)D3 concentrations than those with fair/pale skin and multivariate genetic analysis showed that approximately half of the total additive genetic variation in 25(OH)D3 results from genes whose primary influence is on skin colour and sun exposure. Additionally, 37% of the total variance was attributed to shared environmental effects on vitamin D, skin colour and sun exposure measures. These results support a moderate estimate of vitamin D heritability and suggest significant influence of season, skin colour and sun exposure on the genetic variance.

Cost-Effective HPLC-UV Method for Quantification of Vitamin D2 and D3 in Dried Blood Spot: A Potential Adjunct to Newborn Screening for Prophylaxis of Intractable Paediatric Seizures.

25-Hydroxyvitamin D (25-(OH)D) deficiency is recently been described as one of the multiple factors responsible for pediatric seizures. 25-Hydroxyvitamin D3 and 25-Hydroxyvitamin D2 are the well-known markers to determine Vitamin D status. In this work we report the development of a sensitive and cost effective HPLC technique for the quantification of the vitamin D metabolites from dried blood spot samples (DBS). The metabolites were extracted using acetonitrile-methanol-0.1% formic acid (60 : 20 : 20 (v/v)) and analyzed on an Acclaim C18 column (150 × 4.6 mm i.d., 3 µm) at a flow rate of 1 mL/min. The method was linear in the range of 10-80 ng/mL. Limit of detection and limit of quantification (LOQ) of the method were 5 and 10 ng/mL respectively. Extensive stability studies demonstrated the analytes to be stable in stock and matrix with a percent change within the acceptable range of ±15%. Comparison of the newly developed HPLC-DBS method with the reported LC-MS-DBS and electrochemiluminescence immunoassay (ECLIA) methods followed by Bland-Altman analysis demonstrated a bias of 0.08 and -0.14, respectively proving the methods are comparable. Application of the developed method to a pediatric seizure cohort depicted 46.6% of cases as deficient and 26.6% as insufficient for 25-(OH)D. Among deficient cases 8 samples were below 10 ng/mL and exact amount was not calculated since these were below the LOQ levels. The mean ± standard deviation (S.D.) in the remaining 6 deficient cases was 13.22 ± 2.80 ng/mL. The levels in healthy infants were 33.9 ± 6.11 ng/mL. The method can be used routinely for assessing 25-(OH)D deficiency in newborn.

Vitamin D Content of Australian Native Food Plants and Australian-Grown Edible Seaweed.

Vitamin D has previously been quantified in some plants and algae, particularly in leaves of the Solanaceae family. We measured the vitamin D content of Australian native food plants and Australian-grown edible seaweed. Using liquid chromatography with triple quadrupole mass spectrometry, 13 samples (including leaf, fruit, and seed) were analyzed in duplicate for vitamin D2, vitamin D3, 25-hydroxyvitamin D2, and 25-hydroxyvitamin D3. Five samples contained vitamin D2: raw wattleseed (Acacia victoriae) (0.03 µg/100 g dry weight (DW)); fresh and dried lemon myrtle (Backhousia citriodora) leaves (0.03 and 0.24 µg/100 g DW, respectively); and dried leaves and berries of Tasmanian mountain pepper (Tasmannia lanceolata) (0.67 and 0.05 µg/100 g DW, respectively). Fresh kombu (Lessonia corrugata) contained vitamin D3 (0.01 µg/100 g DW). Detected amounts were low; however, it is possible that exposure to ultraviolet radiation may increase the vitamin D content of plants and algae if vitamin D precursors are present.

Effect of pasteurisation on the concentrations of vitamin D compounds in donor breastmilk.

Premature infants are the main recipients of pasteurised donor human milk (PDHM), when their mothers are unable to provide their own. In this study, we evaluated the effect of pasteurisation on the concentrations of vitamin D compounds in donor breastmilk. Milk samples were obtained pre- and post-Holder pasteurisation. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to analyse the samples for vitamins D2 and D3 (D2 and D3) and 25-hydroxyvitamins D2 and D3 (25(OH)D2 and 25(OH)D3). The significance of differences in vitamin D concentrations between the two groups of milk samples was assessed using the Wilcoxon matched-pairs signed rank test, in which p < 0.05 was considered significant. Pasteurisation resulted in a significant reduction (p < 0.05) in the content of D2, D3, 25(OH)D2 and 25(OH)D3. The losses ranged from 10% to 20% following pasteurisation.

Determination of four sulfated vitamin D compounds in human biological fluids by liquid chromatography-tandem mass spectrometry.

The determination of both the water-soluble and lipid-soluble vitamin D compounds in human biological fluids is necessary to illuminate potentially significant biochemical mechanisms. The lack of analytical methods to quantify the water-soluble forms precludes studies on their role and biological functions; currently available liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods are able to determine only a single sulfated form of Vitamin D. We describe here a highly sensitive and specific LC-MS/MS method for the quantification of four sulfated forms of vitamin D: vitamins D2- and D3-sulfate (D2-S and D3-S) and 25-hydroxyvitamin D2- and D3-sulfate (25(OH)D2-S and 25(OH)D3-S). A comparative evaluation showed that the ionization efficiencies of underivatized forms in negative ion mode electrospray ionisation (ESI) are superior to those of the derivatized (using 4-phenyl-l,2,4-triazoline-3,5-dione (PTAD)) forms in positive ion mode ESI. Separation was optimised to minimise co-elution with endogenous matrix compounds, thereby reducing ion suppression/enhancement effects. Isotopically labelled analogues of each compound were used as internal standards to correct for ion suppression/enhancement effects. The method was validated and then applied for the analysis of breastmilk and human serum. The detection limits, repeatability standard deviations, and recoveries ranged from 0.20 to 0.28fmol, 2.8 to 10.2%, and 81.1 to 102%, respectively.

Vitamin D activity of breast milk in women randomly assigned to vitamin D3 supplementation during pregnancy.

BACKGROUND: Human milk is typically low in vitamin D activity (VDA). Whether the vitamin D content of breast milk at birth can be increased by supplementing the mother during pregnancy has not been reported to the best of our knowledge. OBJECTIVE: We examined the effect of vitamin D supplementation during pregnancy on breast-milk VDA in the first 2 mo of lactation. DESIGN: Breast-milk samples were obtained from women who were enrolled in a randomized, double-blinded, placebo-controlled trial of vitamin D supplementation during pregnancy. Pregnant women were enrolled at 27 wk of gestation and randomly assigned to the following 3 groups: a placebo group, a group who received one dosage of daily oral vitamin D3 (1000 IU), or a group who received 2 dosages of daily oral vitamin D3 (2000 IU). Serum 25-hydroxyvitamin D [25(OH)D] was measured at enrollment, at 36 wk of gestation, and in cord blood at birth. Study participants who were breastfeeding were invited to provide breast-milk samples for VDA measurement [concentration of vitamin D2, vitamin D3, 25(OH)D2, and 25(OH)D3] at 2 wk and 2 mo postpartum. A linear mixed model was used to compare breast-milk VDA between the 3 study groups. RESULTS: A total of 75 women provided breast-milk samples (44 women provided breast-milk samples at both 2 wk and 2 mo postpartum). The mean (95% CI) VDA at age 2 wk was 52 IU/L (12, 217 IU/L) in the placebo group, 51 IU/L (17, 151 IU/L) in the 1000-IU group, and 74 IU/L (25, 221 IU/L) in the 2000-IU group; and at age 2 mo, the mean (95% CI) VDA was 45 IU/L (16, 124 IU/L), 43 IU/L (18, 103 IU/L), and 58 IU/L (15, 224 IU/L), respectively. There was no significant interaction in VDA between the sample-collection time and treatment (P = 0.61), but there was a difference between lower- and higher-dosage treatment groups (P = 0.04). CONCLUSION: Maternal vitamin D supplementation during pregnancy of 2000 IU/d (compared with 1000 IU/d and with a placebo) results in a higher VDA of breast milk ≥2 mo postpartum. This trial was registered at the Australian New Zealand Clinical Trials Registry as ACTRN12610000483055.

Vitamina D, determinante de la salud ósea y extra ósea; importancia de su suplementación en la leche y derivados / Vitamin D, determinant of bone and extrabone health. Importance of Vitamin D supplementation in milk and dairy products

La vitamina D se obtiene fundamentalmente a partir de la irradiación ultravioleta en la piel del 7-dehidrocolesterol para formar colecalciferol (vitamina D3) y mínimamente por la dieta, salvo que se tomen alimentos fortificados en vitamina D, fundamentalmente leche; en algunos países se emplea ergocalciferol (vitamina D2). En el hígado la vitamina D3 se hidroxila para formar 25-hidroxivitamina D3 (marcador del estatus nutricional corporal en vitamina D). La 25OHD3, se hidroxila para formar 1,25-dihidroxivitamina D3 (1,25OH)2D3 en el riñón, para controlar la homeostasis del calcio y la salud del hueso y en otras células o tejidos, mediante el estímulo del VDR, incluyendo piel, músculo, los sistemas cardiovascular e inmune, homeostasis de la glucosa, y proliferación celular en general; de tal manera, que alrededor del 3% del genoma humano está regulado por la hormona 1,25(OH)2 vitamina D3. Estudios de asociación describen acciones beneficiosas a nivel cardiovascular, hipertensión arterial, cáncer colorectal, de mama, esclerosis múltiple, función inmune e inflamación etc. Un objetivo mínimo irrenunciable, para la salud pública, debe ser conseguir niveles séricos de 25OHD superiores a 20 ng/ml, para asegurar un estatus óptimo para la salud ósea y preferiblemente mayor de 30 ng/ml, si nos proponemos alcanzar otros objetivos. 'Paradójicamente' en España se da una elevada prevalencia de insuficiencia o incluso franca deficiencia de vitamina D en niños y jóvenes, persiste en adultos, en mujeres postmenopáusicas (osteoporóticas o no), o ancianos que viven en sus casas, y que es mayor si viven en residencias, con una variación estacional que apenas llega a normalizar los niveles séricos de 25OHD después del verano-otoño. También se ha demostrado una elevada prevalencia de niveles inadecuados de vitamina D en mujeres posmenopáusicas en tratamiento por osteoporosis con niveles de 25-hidroxivitamina D menores de 30 ng/ml y 20 ng/ml en el 63 y 30% respectivamente, lo que constituye un importante factor contribuyente a falta respuesta ósea al tratamiento. Una adecuación de niveles séricos de vitamina D, permitiría que la dieta proporcionara el calcio necesario para conseguir una buena salud ósea. Dada la dificultad para conseguir niveles adecuados de vitamina D por irradiación UV y por dieta, la suplementación adecuada de leche y derivados con vitamina D supone una atractiva posibilidad y un reto, para la Salud Pública de España y la Unión Europea, que ha dado excelentes resultados en EEUU, Canadá, Países de Norte de Europa, etc (AU)

Vitamin D is obtained mainly from ultraviolet irradiation of 7-dehydrocholesterol in the skin to form cholecalciferol (vitamin D3), and minimally from diet, unless vitamin D fortified food is taken, mainly enriched milk. In some countries, vitamin D is added to diet as ergocalciferol (vitamin D2). In the liver, vitamin D3 is hydroxylated to form 25-hydroxyvitamin D3 (marker of body nutritional status of vitamin D). Subsequently, in the kidney, 25OHD3 is hydroxylated to form 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). By VDR stimulation, (1,25)OH)2D3 controls calcium homeostasis and bone health and, what is more, many other cells and tissues including skin, muscle, cardiovascular and immune systems as well as glucose homeostasis. Thus, about 3% of the human genome is regulated by this hormone. Association and recent intervention studies describe beneficial effects on bone, cardiovascular disease, hypertension, diabetes mellitus type 2,colorectal cancer, breast cancer, multiple sclerosis, immune function inflammation etc. A minimum target for public health should be to achieve serum 25OHD levels above 20 ng/ml to ensure optimum status for bone health. However, levels above 30 ng/ml should be reached to achieve other health goals. Paradoxically, inadequacy (or even deficiency) in vitamin D levels is highly prevalent in children and youth in Spain. This deficit persists in adults, as well as in postmenopausal women (osteoporotic or not) and the elderly (especially amongst those institutionalized). Seasonal variation barely normalizes serum 25OHD levels after summer-autumn. Treated postmenopausal osteoporotic women also show high prevalence of inadequate levels of vitamin D, a major contributor to antiresortive treatments failure. A normalization of serum vitamin D enables diet to provide the calcium necessary to achieve a good bone health and an adequate response to antiresortive drugs. Given the difficulty to get adequate levels of vitamin D by UV irradiation and diet, a proper supplementation of milk with vitamin D is an attractive chance and a challenge for Public Health of Spain and the European Union. It has provided excellent results in the US, Canada, Northern Europe Countries, etc (AU)

Overreporting of vitamin D deficiency with the Roche Elecsys Vitamin D3 (25-OH) method.

BACKGROUND: Vitamin D deficiency is common. Recently Roche Diagnostics removed their Elecsys Vitamin D3 (25OH) electrochemiluminescence immunoassay (ECLIA) from use, citing deteriorating traceability to the reference method (liquid chromatography tandem mass spectrometry; LCMSMS). We investigated the performance of the Roche assay (2 assay formulations) against an LCMSMS method and the widely used DiaSorin radioimmunoassay (RIA) method. METHODS: Two sets of samples from separate populations were assayed for vitamin D. The first set was assayed using three different methods: RIA (DiaSorin) in 2004, polyclonal ECLIA (Roche) in early 2009 and LCMSMS in early 2010. The second set was assayed using polyclonal and monoclonal ECLIA (Roche) and LCMSMS in mid-2010. RESULTS: The correlation of the polyclonal ECLIA with the RIA was poor (ECLIA = 0.45 × RIA + 19, r(2) = 0.59, n = 773). LCMSMS results correlated with RIA (RIA = 0.86 × LCMSMS + 4, r(2) = 0.69, n = 49) better than with polyclonal ECLIA (polyclonal ECLIA = 0.55 × LCMSMS + 6, r(2) = 0.62, n = 55) despite a storage interval of 6 years.In recently collected samples monoclonal and polyclonal immunoassays gave similar results (monoclonal ECLIA = 0.93 polyclonal ECLIA -3, r(2) = 0.60, n = 153). The correlation between monoclonal Roche ECLIA and LCMSMS in these samples was very poor (monoclonal ECLIA = 0.31 × LCMSMS + 23, r(2) = 0.27). CONCLUSIONS: At the time of its removal from the market, the Roche Elecsys Vitamin D3 (25OH) assay showed unacceptable performance, underestimating vitamin D levels. It seems that this bias preceded the introduction of the monoclonal assay. The worldwide distribution of the assay and the duration of this bias likely led to a significant number of patients starting supplementation unnecessarily.

Development and optimization of an LC-MS/MS-based method for simultaneous quantification of vitamin D2 , vitamin D3 , 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3.

Simultaneous and accurate measurement of vitamin D and 25-hydroxyvitamin D in biological samples is a barrier limiting our ability to define "optimal" vitamin D status. Thus, our goal was to optimize conditions and evaluate an LC-MS method for simultaneous detection and quantification of vitamin D(2) , vitamin D(3) , 25-hydroxyvitamin D(2) and 25-hydroxyvitamin D(3) in serum. Extraction and separation of vitamin D forms were achieved using acetone liquid-liquid extraction and by a reversed phase C8 column, respectively. Detection was performed on a triple quadrupole tandem mass spectrometer (QQQ-MS/MS) equipped with atmospheric pressure photo ionization source. The LOQs for all analytes tested were 1 ng/mL for hydroxylated molecules and 2 ng/mL for the parent vitamin Ds. RSD at lower LOQ (2 ng/mL) and in medium (80 ng/mL) and high (200 ng/mL) quality control samples did not exceed 20 and 15% CV, respectively. Accuracy of the method for determination of hydroxylated molecules was also validated using National Institutes of Standards and Technology standard samples and found to be in the range of 90.9-111.2%. In summary, a sensitive and reproducible method is reported for simultaneous quantification of vitamin D(2) , vitamin D(3) , 25-hydroxyvitamin D(2) and 25-hydroxyvitamin D(3) molecules in biological samples.

Characterization of monoglucuronides of vitamin D2 and 25-hydroxyvitamin D2 in rat bile using high-performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry.

The characterization of vitamin D2 3-glucuronide, 25-hydroxyvitamin D2 3-glucuronide and 25-hydroxyvitamin D2 25-glucuronide, biliary metabolites obtained from rats dosed with vitamin D2 and 25-hydroxyvitamin D2 per os, was carried out using HPLC-atmospheric pressure chemical ionization (APCI)-MS. The glucuronide obtained from bile specimens was identified by comparison of its chromatographic behaviour with an authentic sample using HPLC-APCI-MS operating in the negative-ion mode. Methylation of the respective fraction with diazomethane gave the methyl ester, which was also confirmed by HPLC-APCI-MS operating in the positive-ion mode. The (M-H)- and (M + NH4)+ ions were monitored in the selected-ion monitoring mode.