Rapid and sensitive high-performance liquid chromatographic method for simultaneous determination of retinol, α-tocopherol, 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 in human plasma with photodiode-array ultraviolet detection

A new rapid and sensitive high-performance liquid chromatographic method using 0.5 ml of plasma has been developed for the simultaneous determination of retinol (vitamin A), α-tocopherol (vitamin E), 25-hydroxyvitamin D₂ and 25-hydroxyvitamin D₃. The eluate was monitored with a photodiode-array detector with two fixed wavelengths (267 nm for vitamin D, 292 nm for α-tocopherol and retinol). For all compounds, including internal standards, the method provides extraction recoveries greater than 81%. Detection limits were equal to or lower than 1.5 μg/l for the 4 vitamins. Linearity of standards was excellent (r>0.999 in all cases). Intra-day and inter-day precision were generally acceptable; the intra-day-assay C.V. was 7.7 for all compounds and the inter-day-assay C.V. was <9.2% except for the lower concentrations of 25-hydroxyvitamin D₃, 25-hydroxyvitamin D₂ and α-tocopherol (10.8, 11.8 and 11.9, respectively). The important properties of the present method are its ease of use, its rapidity, since sample preparation was achieved in 15 min and all the compounds were eluted in less than 15 min, and its small sample volume required (=0.5 ml), which enables it to be used in pediatric practice.     

Vitamin D and prevention of colorectal cancer

BACKGROUND: Inadequate photosynthesis or oral intake of Vitamin D are associated with high incidence rates of colorectal cancer, but the dose-response relationship has not been adequately studied. METHODS: Dose-response gradients from observational studies of Vitamin D intake and serum 25-hydroxyvitamin D were plotted as trend lines. The point on each linear trend line corresponding to an odds ratio of 0.50 provided the prediagnostic Vitamin D intake or 25-hydroxyvitamin D concentration associated with 50% lower risk compared to <100IU/day Vitamin D or <13ng/ml serum 25-hydroxyvitamin D. Medians of these values were determined. RESULTS: Overall, individuals with >or=1000IU/day oral Vitamin D (p<0.0001) or >or=33ng/ml (82nmol/l) serum 25-hydroxyvitamin D (p<0.01) had 50% lower incidence of colorectal cancer compared to reference values. CONCLUSIONS: Intake of 1000IU/day of Vitamin D, half the safe upper intake established by the National Academy of Sciences, was associated with 50% lower risk. Serum 25-hydroxyvitamin D of 33ng/ml, which is known to be safe, also was associated with 50% lower risk. Prompt public health action is needed to increase intake of Vitamin D(3) to 1000IU/day, and to raise 25-hydroxyvitamin D by encouraging a modest duration of sunlight exposure.     

Quantification of fat-soluble vitamins in human breast milk by liquid chromatography-tandem mass spectrometry

Sensitive quantification method for fat-soluble vitamins in human breast milk by liquid chromatography-tandem mass spectrometry was developed. Vitamins A, D and E were extracted from 10.0 mL of breast milk after saponifying by basic condition. Vitamin K derivatives were extracted from 3.0 mL of breast milk after lipase treatment. The corresponding stable isotope-labeled compounds were used as internal standards. For the determination of vitamin D compounds, derivatization with a Cookson-type reagent was performed. All fat-soluble vitamins were determined by liquid chromatography-tandem mass spectrometry in the positive ion mode. The detection limits of all analytes were 1-250 pg per 50 microL. The recoveries of fat-soluble vitamins were 91-105%. Inter-assay CV values of each vitamin were 1.9-11.9%. The mean concentrations of retinol, vitamin D3, 25-hydroxyvitamin D3, alpha-tocopherol, phylloquinone and menaquinone-4 were 0.455 microg/mL, 0.088 ng/mL, 0.081 ng/mL, 5.087 microg/mL, 3.771 ng/mL, and 1.795 ng/mL, respectively (n=82). This method makes possible to determine fat-soluble vitamins with a wide range of polarities in human breast milk. The assay may be useful for large-scale studies.     

Studies on the analysis of 25-hydroxyvitamin D3 by isotope-dilution liquid chromatography–tandem mass spectrometry using enzyme-assisted derivatisation

The total serum concentration of 25-hydroxyvitamins D (25-hydroxyvitamin D₃ and 25-hydroxyvitamin D₂) is currently used as an indicator of vitamins D status. Vitamins D insufficiency is claimed to be associated with multiple diseases, thus accurate and precise reference methods for the quantification of 25-hydroxyvitamins D are needed. Here we present a novel enzyme-assisted derivatisation method for the analysis of vitamins D metabolites in adult serum utilising 25-[26,26,26,27,27,27-²H₆]hydroxyvitamin D₃ as the internal standard. Extraction of 25-hydroxyvitamins D from serum is performed with acetonitrile, which is shown to be more efficient than ethanol. Cholesterol oxidase is used to oxidize the 3β-hydroxy group in the vitamins D metabolites followed by derivatisation of the newly formed 3-oxo group with Girard P reagent. 17β-Hydroxysteroid dehydrogenase type 10 is shown to oxidize selectively the 3α-hydroxy group in the 3α-hydroxy epimer of 25-hydroxyvitamin D₃. Quantification is achieved by isotope-dilution liquid chromatography–tandem mass spectrometry. Recovery experiments for 25-hydroxyvitamin D₃ performed on adult human serum give recovery of 102–106%. Furthermore in addition to 25-hydroxyvitamin D₃, 24,25-dihydroxyvitamin D₃ and other uncharacterised dihydroxy metabolites, were detected in adult human serum.     

Effect of Vitamin D supplement use on serum concentrations of total 25OHD levels in elderly women

Vitamin D(2) and D(3) are generally considered equipotent in humans. As Vitamin D(2) supplements are commonly used by elderly in United States, we determined the contribution of 25OHD(2) to the total serum 25OHD levels by HPLC in elderly women who reported taking Vitamin D(2) supplements (n = 56) and also in a group of randomly selected unsupplemented women (n = 60). In addition, we compared the total serum 25OHD measured by HPLC with competitive protein-binding assay (CPBA), a method routinely employed to measure Vitamin D status. A correlation of 0.91 (P < 0.001) was observed between the two methods for the serum total 25OHD measurement. The mean serum 25OHD level in Vitamin D(2) supplemented group was significantly higher than in unsupplemented group measured by HPLC (32 versus 28 ng/ml) and marginally higher measured by CPBA (33 vs. 31 ng/ml). Seventy eight percent of women taking Vitamin D(2) supplements had appreciable amounts of circulating 25OHD(2,) which constituted about 25 percent of their total serum 25OHD. It is also interesting to note that Vitamin D deficiency was less prevalent in elderly women taking Vitamin D(2) supplements (1.8%) compared to women not taking any supplements (12%).     

Observations from the UK Supra-Regional Assay Service laboratory for the measurement of Vitamin D metabolites

As a Supra-Regional Assay Service (SAS) laboratory, we receive samples from all over the UK. Of these some are sent frozen and others by post or courier. We have examined transport and storage conditions to see whether they affect the measurement of Vitamin D metabolites and potentially contribute to the variation in measurement of 25-hydroxyvitamin D (25OHD) seen in the DEQAS scheme. We have also examined the samples received during 2005. We found that different transport and storage conditions did not contribute significantly to the normal variation seen in measuring Vitamin D metabolites (CV% (±S.E.) for stored versus assay controls: 5.1 ± 0.06% versus 4.5 ± 0.04% for 25OHD and 10.8 ± 1.0% versus 12.3 ± 1.0% for 1,25D). A review of the service showed a 240% increase in samples received over the last 5 years. Despite an increased awareness of the need to measure Vitamin D status, in this cross-section of patient samples 92% of Asian and 86% of white patients were found to be Vitamin D-insufficient (<30 ng/ml) and 27% of Asian and 14% of white patients were profoundly deficient (<5 ng/ml) and at risk of bone disease.     

Volumetric dilution, rather than sequestration best explains the low vitamin D status of obesity

Vitamin D status is known to be poor in obese individuals; there is no consensus as to the reason. Cross-sectional study of the relation between serum 25-hydroxyvitamin D (25(OH)D) concentration and body size in the baseline data from unsupplemented adults entering two study cohorts in our research unit, N = 686. Regression analyses of body size variables against serum 25(OH)D concentration, using both linear and hyperbolic models. The fit to a hyperbolic model of 25(OH)D against body weight completely removed the obesity-related component of inter-individual variability in serum 25(OH)D concentration. The hyperbolic fit using total body weight was significantly better than any linear model, and specifically better than any using BMI. Dilution of ingested or cutaneously synthesized vitamin D in the large fat mass of obese patients fully explains their typically low vitamin D status. There is no evidence for sequestration of supplemental or endogenous cholecalciferol. Vitamin D replacement therapy needs to be adjusted for body size if desired serum 25(OH)D concentrations are to be achieved.     

Vitamin D Status: A Different Story in the Very Young versus the Very Old Romanian Patients

BackgroundIn Romania (latitude 48°15’N to 43°40’N), vitamin D supplementation is common practice mostly in infants 0-1 year old. No published information is available regarding epidemiological data on vitamin D status in the Romanian population for a wide age range and geographical territory. In this context, we aimed to evaluate the seasonal and age variation of vitamin D status in a large Romanian population.Methods6631 individuals from across Romania had performed 7544 vitamin D assessments (2012-2014) in a chain of private laboratories. Vitamin D (25-hydroxyvitamin D2 and 25-hydroxyvitamin D3) was measured using High Performance Liquid Chromatography. Vitamin D levels were classified as severe deficiency<10ng/mL, deficiency 10-20ng/mL, insufficiency 21-29ng/mL, sufficiency≥30ng/mL and potentially harmful>100ng/ml.ResultsMale to female ratio was 1:2.9. Age ranged from 0 to 85 years. Mean vitamin D levels increased from April (26.3ng/ml) to September (35.6ng/ml) and decreased from October (33.5ng/ml) to March (24.4 ng/ml). Overall 40% had sufficient vitamin D, while the rest were insufficient 33%, deficient 22%, severely deficient 4% and 1% potentially harmful (of them 81% under 1 year old). Males compared to females showed higher percentages of sufficiency (47% vs. 38%). Children 0- 2 years presented the highest percentage of vitamin D sufficiency (77%). Lowest percentages (21%) of sufficiency were in people 80-84 years.ConclusionIn Romania, suboptimal vitamin D levels are common (59%), especially in older age, wintertime and in women. Vitamin D supplementation would be most warranted from January to April in the Romanian population. 25-hydroxyvitamin D levels>100ng/ml were relatively prevalent in children 0-1 year old (17.3%). This was attributed to supplementation errors and the fact that high-risk individuals were more likely to visit for medical check-up. Nonetheless, it stresses the need to increase awareness of the importance of preventing Vitamin D supplementation administration errors in the young.     

The Vitamin D requirement in health and disease

Advances in Vitamin D nutritional physiology since publication of the DRIs in 1997 are briefly summarized. Available data indicate that (1) Vitamin D's canonical function, optimizing intestinal calcium absorption, is fully expressed at serum 25-hydroxyvitamin D (25OHD) concentration of approximately 80nmol/L; (2) elevated parathyroid activity, typical of aging populations, is minimized at the same 25OHD value and (3) osteoporotic fractures are reduced when serum 25OHD is raised to near 80nmol/L. Depending upon starting value, achieving 25OHD concentrations of 80 or higher may require a daily oral intake of 2200IU (55microg) or more in addition to prevailing cutaneous inputs. The tolerable upper intake level (TUIL), currently set at 2000IU (50microg)/day, is too low to permit optimization of Vitamin D status in the general population. Actual toxicity is not seen below serum 25OHD values of 250nmol/L, a value that would be produced only at continuing oral intakes in excess of 10,000IU (250microg)/day.     

Subcellular localization of vitamin D3 25-hydroxylase in human liver

Vitamin D3 25-hydroxylase activity was measured in subcellular and submitochondrial fractions of human liver. Quantitation of 25-hydroxyvitamin D3 was based on high performance liquid chromatography. Vitamin D3 25-hydroxylase activity was detected in the mitochondrial fraction only. The mitochondrial 25-hydroxylase activity was linear with time up to 60 min and with mitochondrial protein up to 1 mg/ml. An apparent Km value of about 10(-5) M was found. Substrate satuation level was not reached. In the presence of 2.4 X 10(-4) M vitamin D3, the rate of 25-hydroxyvitamin D3 formation was 0.19 nmol X mg of protein-1 X h-1 After fractionation of the mitochondria, 86% of the 25-hydroxylase activity was recovered in the mitoplast fraction. The outer membrane fraction was devoid of activity. It is concluded that human liver contains only one detectable vitamin D3 25-hydroxylase enzyme localized to the mitochondrial inner membrane.     

Which circulating level of 25-hydroxyvitamin D is appropriate?

Moderate Vitamin D deficiency causes secondary hyperparathyroidism and bone loss, leading to osteoporosis and fractures. Controversy exists which circulating level of 25-hydroxyvitamin D (25OH)D is appropriate. The high incidence of hip fractures at northern latitudes suggest a relationship with Vitamin D deficiency. However, international studies show lower serum 25(OH)D levels in southern than in northern Europe. Serum 25(OH)D was not a risk factor for hip fractures in several epidemiological studies. The required serum 25(OH)D is usually established by assessing the point where serum parathyroid hormone (PTH) starts to rise. This point varied in several studies between 30 and 78 nmol/l. However, interlaboratory variation may also influence the apparent required serum 25(OH)D level. Dietary calcium intake influences serum PTH and serum PTH may influence the turnover of Vitamin D metabolites. A low calcium intake causes an increase of serum PTH and serum 1,25(OH)2D thereby decreasing the half life of serum 25(OH)D. While a low calcium intake may aggravate Vitamin D deficiency, a high calcium intake may have a Vitamin D sparing effect. With current knowledge, a global estimate for the appropriate serum 25(OH)D is 50 nmol/l.     

孕妇维生素D缺乏与妊娠期糖尿病的相关性分析

目的:调查孕妇妊娠早期维生素D水平及其影响因素,探讨维生素D缺乏与妊娠期糖尿病的相关性。方法:选取2012年7月至2013年4月在上海交通大学医学院附属新华医院产科正规产检并分娩的非孕前糖尿病孕妇,在其建卡初检时采用电化学发光免疫技术测定血清25(OH)D3水平;妊娠24-28周行糖筛查及糖耐量试验,诊断是否为妊娠期糖尿病GDM。收集并整理孕妇年龄,孕前体重指数BMI、维生素D测定孕周与测定季节、孕期维生素D补充情况等信息。结果:1000例孕妇中,GDM发病率为11.5%,维生素D缺乏比例占67.4%;其中,约有54%孕妇常规补充复合维生素,约含维生素400 IU/天,10%孕妇常规补充维生素D。GDM孕妇25(OH)D3水平显著低于正常对照组(P=0.007)。维生素D缺乏孕妇发生GDM的风险是维生素D水平较高组的1.944倍,且在秋冬季更易发生GDM。可以考虑在孕14-16周进行维生素D水平的早期测定。结论:孕妇维生素D缺乏十分普遍。妊娠早期孕妇低维生素D水平可能增加孕妇胰岛素抵抗及孕期发生GDM的发生风险。     

Vitamin D and prevention of breast cancer: pooled analysis

BACKGROUND: Inadequate photosynthesis or oral intake of Vitamin D are associated with high incidence and mortality rates of breast cancer in ecological and observational studies, but the dose-response relationship in individuals has not been adequately studied. METHODS: A literature search for all studies that reported risk by of breast cancer by quantiles of 25(OH)D identified two studies with 1760 individuals. Data were pooled to assess the dose-response association between serum 25(OH)D and risk of breast cancer. RESULTS: The medians of the pooled quintiles of serum 25(OH)D were 6, 18, 29, 37 and 48 ng/ml. Pooled odds ratios for breast cancer from lowest to highest quintile, were 1.00, 0.90, 0.70, 0.70 and 0.50 (p trend<0.001). According to the pooled analysis, individuals with serum 25(OH)D of approximately 52 ng/ml had 50% lower risk of breast cancer than those with serum <13 ng/ml. This serum level corresponds to intake of 4000 IU/day. This exceeds the National Academy of Sciences upper limit of 2000 IU/day. A 25(OH)D level of 52 ng/ml could be maintained by intake of 2000 IU/day and, when appropriate, about 12 min/day in the sun, equivalent to oral intake of 3000 IU of Vitamin D(3). CONCLUSIONS: Intake of 2000 IU/day of Vitamin D(3), and, when possible, very moderate exposure to sunlight, could raise serum 25(OH)D to 52 ng/ml, a level associated with reduction by 50% in incidence of breast cancer, according to observational studies.     

Measurement of Vitamin D metabolites: an international perspective on methodology and clinical interpretation

The International Quality Assessment Scheme for Vitamin D metabolites (DEQAS) was introduced in 1989. Initially, the aim was to improve the reliability of 25-hydroxyvitamin D (25-OHD) assays but the scheme was extended in 1997 to include 1,25-dihydroxyvitamin D (1,25(OH)(2)D). DEQAS has 95 members in 18 countries (January 2003). Five serum samples are distributed quarterly and participants are given up to 6 weeks to return their results for statistical analysis. The majority of participants use commercial kits for both analytes. A performance target was set by an advisory panel in 1997 and, at present, requires participants to get 80% or more of their results within +/-30% of the All-Laboratory Trimmed Mean (ALTM). The performance targets are under continual review. In 2003, 59% of participants met the target (cf. 52% in 2000). A questionnaire, distributed in January 2003, requested information on methods and the interpretation of results. Reference ranges varied but there was reasonable agreement on the 25-OHD concentrations below which Vitamin D supplementation was advised. A minority (22%) of respondents was unsure whether Vitamin D(3) or Vitamin D(2) was used to treat patients in their locality. The majority (52%) of assays for 1,25(OH)(2)D were done 'on demand' and others for apparently spurious reasons. Most respondents thought participation in DEQAS extremely important and the planned introduction of on-line reporting should enhance its value.     

Vitamin D deficiency as a risk factor for urinary tract infection in women at reproductive age

Vitamin D deficiency is a pandemic problem and an ever-increasing issue in human nutrition and health. Vitamin D (serum 25-hydroxyvitamin D) deficiency causes many health problems such as autoimmune diseases, Crohn’s disease, diabetes, inflammation, asthma, hypertension, and cancer. Vitamin D3 (cholecalciferol) deficiency has been documented as a persistent problem among adults, children, and elderly persons in most of the countries. Our main objective of this study was to determine the hypothesis that the vitamin D deficiency among women can lead to them developing frequent urinary tract infections. Vitamin D has a potential role in immune regulation and it prevents infections especially urinary tract infections (UTI). Therefore it has positive regulatory role in both acute and recurrent infections especially in women of reproductive ages. As women at this age group have specific differences in their urinary tract and the reproductive organ anatomy, make them more prone for micro-organisms' invasion, The present study was carried out to ascertain certain relation between serum 25-hydroxyvitamin D levels and UTI in women while contemplating the significance of knowing the risk factors associated with UTI and also finding ways to avoid serious complications. 75 women with (case group) UTI were differentiated with 35 healthy with no UTI (control group) and 40 women with UTI and their serum 25-hydroxyvitamin D levels were checked in a case control study. The women were between at 17–52 years of age. Using ELISA, Serum 25-hydroxyvitamin D levels were measured. Analysis and comparison of the results were done among the two groups. Vitamin D mean levels in the case group was considerably lower when in comparison with the control group (11.09 ± 7.571 ng/mL vs. 24.08 ± 11.95 ng/mL, P < 0.001).     

A prospective randomized controlled trial of the effects of vitamin D supplementation on cardiovascular disease risk

Vitamin D (VitD) supplementation has been advocated for cardiovascular risk reduction; however, supporting data are sparse. The objective of this study was to determine whether VitD supplementation reduces cardiovascular risk. Subjects in this prospective, randomized, double-blind, placebo-controlled trial of post-menopausal women with serum 25-hydroxyvitamin D concentrations >10 and <60 ng/mL were randomized to Vitamin D3 2500 IU or placebo, daily for 4 months. Primary endpoints were changes in brachial artery flow-mediated vasodilation (FMD), carotid-femoral pulse wave velocity (PWV), and aortic augmentation index (AIx). The 114 subjects were mean (standard deviation) 63.9 (3.0) years old with a 25-hydroxyvitamin D level of 31.3 (10.6) ng/mL. Low VitD (<30 ng/mL) was present in 47% and was associated with higher body-mass index, systolic blood pressure, glucose, CRP, and lower FMD (all p<0.05). After 4 months, 25-hydroxyvitamin D levels increased by 15.7 (9.3) ng/mL on vitamin D3 vs. -0.2 (6.1) ng/mL on placebo (p<0.001). There were no significant differences between groups in changes in FMD (0.3 [3.4] vs. 0.3 [2.6] %, p = 0.77), PWV (0.00 [1.06] vs. 0.05 [0.92] m/s, p = 0.65), AIx (2.7 [6.3] vs. 0.9 [5.6] %, p = 0.10), or CRP (0.3 [1.9] vs. 0.3 [4.2] mg/L, p = 0.97). Multivariable models showed no significant interactions between treatment group and low VitD status (<30 ng/mL) for changes in FMD (p = 0.65), PWV (p = 0.93), AIx (p = 0.97), or CRP (p = 0.26). In conclusion, VitD supplementation did not improve endothelial function, arterial stiffness, or inflammation. These observations do not support use of VitD supplementation to reduce cardiovascular disease risk.     

Isolation and identification of 25-hydroxyvitamin D3-26,23-peroxylactone. A novel in vivo metabolite of vitamin D3

A new vitamin D3 metabolite was isolated in pure form (18.2 micrograms) from the serum of rats given large doses (two doses of 26 mumol/rat) of vitamin D3. The new metabolite has been unequivocally identified as 3 beta, 25-dihydroxy-9,10-seco-5,7,10(19)-cholestatrieno-26,23-peroxylactone by ultraviolet absorption spectrophotometry, Fourier transform infrared spectrophotometry, mass spectrometry, field desorption mass spectrometry, and specific chemical reaction with triphenyl phosphine. The stereochemical configuration at the C-23 and c-25 positions of the 25-hydroxyvitamin D3-26-23-peroxylactone was definitely determined to be the 23(S)25(R),25-hydroxyvitamin D3-26,23-peroxylactone is suggested for this metabolite. The isolation involved chloroform-methanol extraction and four column chromatographic procedures. The metabolite purification and elution position on these columns were followed by UV measurement at 264 nm. This metabolite was ultimately resolved from the previously known 25-hydroxyvitamin D3-26,23-lactone by high pressure liquid chromatography using a Zorbax Sil column. The 25-hydroxyvitamin D3-26,23-peroxylactone was converted upon storage at room temperature or -20 degrees C into the 25-hydroxyvitamin D3-26,23-lactone. Since under the conditions of this isolation only the 26,23-peroxylactone and no 26,23-lactone of 25-hydroxyvitamin D3 was present in the rat serum, this suggests that the 25-hydroxyvitamin D3-26,23-peroxylactone is the naturally occurring metabolite.     

Relationship of Serum Vitamin D Concentrations and Allostatic Load as a Measure of Cumulative Biological Risk among the US Population: A Cross-Sectional Study

IntroductionThe allostatic load (AL) index is a multi-systemic measure of physiologic dysregulation known to be associated with chronic exposure to stress and adverse health outcomes. We examined the relationship between AL and serum 25-hydroxyvitamin D (25(OH)D) concentration in non-institutionalized US adults.MethodsData from the Third National Health and Nutrition Examination Survey (NHANES III, 1988–94) were used to calculate two versions of AL including 9 biomarkers and another two with 14 biomarkers (systolic and diastolic blood pressure, pulse rate, serum cholesterol, serum HDL-cholesterol, glycated hemoglobin, sex-specific waist-to-hip ratio, serum albumin, and serum C-reactive protein for AL1, and, additionally body mass index, serum triglyceride, serum creatinine, and serum herpes I & II antibodies for AL2), each set defined by predefined cut-offs or by quartiles. Serum vitamin D concentration was ranked into quartiles. Logistic regression, Poisson regression and linear regression were used to examine the association of serum 25(OH)D concentrations on AL, after adjusting for biological, physiological, socioeconomic, lifestyle, and health variables.ResultsOdds Ratios (OR) for high AL of the lowest 25(OH)D serum quartile were between 1.45 (95% CI: 1.28, 1.67) and 1.79 (95% CI: 1.39, 2.32) for the fully adjusted model, depending on AL version. Inverse relationships between vitamin D serum concentrations were observed for all AL versions and every adjustment. This relationship was consistent after stratification by sex, age or ethnic background. Sensitivity to low 25(OH)D concentrations was highest among the youngest group (20–39 years) with an OR of 2.11 (95% CI: 1.63, 2.73) for the lowest vitamin D quartile Q1.ConclusionsVitamin D had a consistent and statistically significant inverse association with all tested models of high AL, which remained consistent after adjusting for biological, socioeconomic, lifestyle and health variables. Our study adds evidence linking low 25(OH)D concentrations with poorer health, further-reaching than bone health.     

Polyclonal antibodies to EB1089 (seocalcitol), an analog of 1alpha,25-dihydroxyvitamin D(3)*

A hapten derivative of EB1089 [1(R),3(S),25-trihydroxy-26,27-dimethyl-9,10-seco-24-homocholesta-5(Z),7(E),10(19),22(E),24(E)-pentaene], a side-chain analog of 1alpha,25-dihydroxyvitamin D(3), was synthesized for raising antibodies with a high specificity for EB1089. The A-ring moiety of EB1089 was replaced in the hapten by a linker for conjugation to a protein. Three polyclonal antibodies were obtained by immunizing rabbits with a BSA-conjugate of the hapten. The antibodies were characterized for titer, avidity and specificity using an enzyme immunoassay with covalently bound EB1089. The three antibodies had similar binding profiles and were highly selective for EB1089 and its metabolites over the naturally occurring vitamin D metabolites. Cross-reactivities with 25-hydroxyvitamin D(3), the most abundant vitamin D metabolite in serum, were in the range 0.01-0.2% relative to EB1089.