Determination of vitamin D3 and 25-hydroxyvitamin D3 in foodstuffs by HPLC UV-DAD and LC–MS/MS

In order to generate new data for vitamin D content for the Canadian Nutrient File, a method for the quantification of vitamin D₃ and 25(OH)D₃ in foodstuffs has been modified and improved. Vitamin D₃ was quantified using reverse phase liquid chromatography (LC) with UV-diode array detector (UV-DAD), while 25(OH)D₃ was measured by triple quadrupole mass spectrometry (APCI MS/MS). Quantification was by internal standards (IS) using vitamin D₂ and 25(OH)D₂. A Certified Reference Material (CRM-421 containing vitamin D₃) and a control sample (internally generated reference material of ground pork containing both vitamin D₃ and 25(OH)D₃) were used as validation and quality control tools. Limit of detection for both compounds was 0.04 μg/100 g. Accuracy for vitamin D in the CRM-421 was 99% (0.142 mg/kg for a target of 0.143, n = 10). Recovery of vitamin D₃ in ground pork was 97% (88% absolute recovery). For 25(OH)D₃, a recovery of 94% (73% absolute recovery) was obtained. Using this method, data for vitamin D₃ and 25(OH)D₃ content in a variety of foods (pork, beef, eggs, poultry, fish, and dinners) have been generated.     

Relative concentrations of 25-hydroxyvitamin D2/D3 and 1,25-dihydroxyvitamin D2/D3 in maternal plasma at delivery

Quantitation of circulating 25-OH-D₂, 25-OH-D₃, 1,25-(OH)₂-D₂ and 1,25-(OH)₂-D₃ was performed on the plasma from 10 women collected at delivery following full term pregnancies. These data indicate that approximately 50% of the circulating 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D in these women are in the vitamin D₂ form. Further, they demonstrate that vitamin D₂ contributes significantly to the total vitamin D status of these individuals and cannot be considered of trivial importance. It is apparent from this study that assay techniques which specifically quantitate vitamin D₂, as well as D₃ must be employed when measuring the total vitamin D status of individuals who are consuming significant quantities of dietary vitamin D₂.     

Dietary vitamin D intake is not associated with 25-hydroxyvitamin D3 or parathyroid hormone in elderly subjects,whereas the calcium-to-phosphate ratio affects parathyroid hormone

This cross-sectional study investigates whether serum 25-hydroxyvitamin D₃ [25(OH)D₃] and intact parathyroid hormone (iPTH) are affected by vitamin D, calcium, or phosphate intake in 140 independently living elderly subjects from Germany (99 women and 41 men; age, 66-96 years). We hypothesized that habitual dietary intakes of vitamin D, calcium, and phosphate are not associated with 25(OH)D₃ or iPTH and that body mass index confounds these associations. Serum 25(OH)D₃ and iPTH were measured by an electrochemiluminescence immunoassay. Dietary intake was determined using a 3-day estimated dietary record. The median dietary intake levels of vitamin D, calcium, and phosphate were 3 μg/d, 999 mg/d, and 1250 mg/d, respectively. Multiple regression analyses confirmed that dietary vitamin D and calcium did not affect 25(OH)D₃ or iPTH; however, supplemental intakes of vitamin D and calcium were associated with 25(OH)D₃ after adjustment for age, sex, body composition, sun exposure, physical activity, and smoking. In addition, phosphate intake and the calcium-to-phosphate ratio were associated with iPTH after multiple adjustments. In a subgroup analysis, calcium and vitamin D supplements, as well as phosphate intake, were associated with 25(OH)D₃ and/or iPTH in normal-weight subjects only. Our results indicate that habitual dietary vitamin D and calcium intakes have no independent effects on 25(OH)D₃ or iPTH in elderly subjects without vitamin D deficiency, whereas phosphate intake and the calcium-to-phosphate ratio affect iPTH. However, vitamin D and calcium supplements may increase 25(OH)D₃ and decrease iPTH, even during the summer, but the impact of supplements may depend on body mass index.     

液相色谱-串联质谱法检测人血清25羟维生素D_3水平及条件控制分析

目的人血清25羟维生素D3[25-(OH)D3]测定对于评价体内维生素D的水平十分重要。本文拟建立液相色谱-串联质谱法(LC-MS/MS)测定人血清25-(OH)D3的方法。方法人血清样本(400μl)用纯甲醇去蛋白,离心后用正己烷进行液-液萃取。采用大气压化学离子源(APCI源),正离子多反应监测模式。色谱柱:Waters ACQUITY UPLC BEH C18色谱柱(50mm×2.1mm,1.7μm);流动相:100%甲醇;流速:0.2ml/min。结果 LC-MS/MS的检出限为1.0ng/ml,25-OHD3浓度在6～120ng/ml范围内,峰面积与浓度线性关系良好(r=0.999),平均回收率为99.5%(范围:97.1%～103.4%),RSD为4.7%。分别用LC-MS/MS和放射免疫法检测53例血清样本,两种方法的平均浓度分别为23.69ng/ml和20.1ng/ml,LC-MS/MS比放射免疫法(RIA)平均高17.69%(P=0.083)。二者的相关性良好,RIA=LC-MS/MS-3.57,相关系数为0.883。结论本方法具有良好的灵敏度、准确度、精确度,易于操作,与放射免疫法相关性好,可用于测定人血清中25-(OH)D3的含量。     

Association of Dietary Vitamin D Intake,Serum 25(OH)D3, 25(OH)D2 with Cognitive Performance in the Elderly

Background: As life expectancy increases, cognitive performance decline in the elderly has become one of the major global challenges. We aimed to evaluate the association of dietary vitamin D (VD), serum 25-hydroxyvitamin D3 (25(OH)D₃), 25-hydroxyvitamin D2 (25(OH)D₂), and total 25-hydroxyvitamin (25(OH)D) concentration with cognitive performance in older Americans. Methods: The data from the National Health and Nutrition Examination Survey (NHANES), 2011–2014 was used. The cognitive performance was assessed by the Consortium to Establish a Registry for Alzheimer’s Disease (CERAD) Word Learning sub-test, Animal Fluency test, and Digit Symbol Substitution Test (DSST). A binary logistic regression model was applied to evaluate the association between VD and cognitive performance, and restricted cubic spline model was adopted to evaluate the dose–response relationship. Results: While comparing to the lowest dietary VD intake group, the multivariate adjusted odds ratios (ORs) and 95% confidence intervals (CIs) of the highest dietary VD intake group were 0.51 (0.36–0.72) for the Animal Fluency test score and 0.45 (0.31–0.66) for DSST score, respectively; and those of serum total 25(OH)D and 25(OH)D₃ concentration were 0.68 (0.47–0.97) and 0.62 (0.44–0.86) for DSST score. L-shaped relationships were identified for dietary VD intake, serum total 25(OH)D and 25(OH)D₃ concentration with cognition performance. The associations between dietary VD intake, serum total 25(OH)D and cognitive performance were non-significant when stratified by gender. Conclusions: The study indicates that dietary VD intake, serum total 25(OH)D and 25(OH)D₃ concentration were positively associated with cognitive performance. Further studies are needed to clarify the possible effects of dietary VD intake and serum 25(OH)D₂, 25(OH)D₃ on cognitive performance.     

Concentration of vitamin D2 in white button mushrooms (Agaricus bisporus) exposed to pulsed UV light

Enrichment of vitamin D₂ in Agaricus bisporus white button mushroom (WBM) using continuous UV light needs a longer exposure time, which can lead to discoloration. Using a Xenon pulsed UV light source, the yield of vitamin D₂ was evaluated in freshly harvested button mushrooms and mushroom slices after exposure to 2.5, 3, 6 and 9 pulses of UV light at an average dose of 1.150 J/cm² energy per pulse. Increase in vitamin D₂ content was proportionate to the number of pulses of UV light. There was no difference in the vitamin D₂ content of mushrooms between 200 g and 500 g punnets for the corresponding number of pulses (737 ± 81, 847 ± 38, 1148 ± 182, 1611 ± 444% versus 747 ± 48, 911 ± 35, 1307 ± 109, 2028 ± 181% Daily Value/serving, respectively; P > 0.05). Mushrooms in the top layer showed significantly higher amounts of vitamin D₂ content than those in the bottom layer of a 500 g punnet (657 ± 22, 796 ± 76, 1433 ± 138, 1878 ± 178% versus 129 ± 60, 237 ± 117, 403 ± 35, 830 ± 257% DV/serving, respectively; P < 0.01). A single layer of sliced mushrooms (∼5 mm thick) showed higher amounts of vitamin D₂ content than sliced mushrooms packaged together after pulsed UV light exposure (7882 ± 1074, 6867 ± 944, 10,826 ± 472, 13,001 ± 1635% versus 1221 ± 281, 1293 ± 210, 1598 ± 207, 2018 ± 459% DV/serving, respectively; P < 0.001). Discoloration of whole or sliced mushrooms was not observed. Thus, pulsed UV light provides a highly effective method for increasing vitamin D₂ levels in A. bisporus white button mushroom.     

Intake of 25-Hydroxyvitamin D3 May Reduce the Severity of Upper Respiratory Tract Infection: Post hoc Analysis of a Randomized,Double-Blind,Placebo-Controlled,Parallel Group Comparison Study

To evaluate the effects of 25-hydroxyvitamin D₃ (25OHD) on symptoms at the onset of the upper respiratory tract infection (URTI) in subjects with insufficient or deficient serum 25-hydroxyvitamin D levels, we conducted a post hoc analysis of data from a randomized, placebo-controlled study; the subjects received 10 μg of 25OHD per day or a placebo for 16 weeks. The Wisconsin Upper Respiratory Symptom Survey-21 was used to determine URTI. The study endpoints included WURSS-21 scores, number of URTI events, and proportion of medication (antibiotics, antipyretic analgesics) usage. We found that the physical symptom scores for “Runny nose,” “Sneezing,” and “Head congestion” were significantly lower in the 25OHD group than in the placebo group; for all items except “Breathe easily, “the quality of life” scores were significantly improved in the 25OHD group. There was no significant difference in the number of URTI events or the proportion of medication use between the groups. Collectively, the findings of this study indicate that a sufficient 25OHD intake can reduce physical symptoms at the onset of upper respiratory tract infection, particularly nasal symptoms, and may improve the quality of life at the time of onset.     

Association between Circulating Levels of 25-Hydroxyvitamin D3 and Matrix Metalloproteinase-10 (MMP-10) in Patients with Type 2 Diabetes

Background: Matrix metalloproteinase-10 (MMP-10) levels increase progressively starting from early diabetic kidney disease (DKD) stages. Vitamin D₃ (vitD₃) deficit is associated with a higher risk of diabetic microangiopathy. Reduced MMP-10 expression has been observed after exposure to vitD₃. Aim: to assess how vitD₃ status is related to MMP-10 levels in patients with Type 2 diabetes (T2D). Methods: 256 patients with T2D were included in this cross-sectional study. Demographic, clinical and serum MMP-10 and 25-hydroxyvitamin D₃ (25(OH)D₃) levels were collected from each patient. The association between MMP-10 and (25(OH)D₃) levels was assessed using a correlation analysis and fitting a multivariate linear regression model. Results: Serum MMP-10 levels were inversely correlated with circulating 25(OH)D₃ (rho = −0.25; p < 0.001). In the subgroup analysis this correlation was significant in patients with DKD (rho = −0.28; p = 0.001) and in subjects with vitD₃ deficit (rho = −0.24; p = 0.005). In the regression model adjusted for kidney function, body adiposity, smoking and vitD supplementation MMP-10 levels were 68.7 pg/mL lower in patients with 25(OH)D₃ > 20 ng/mL, with respect to ≤20 ng/mL (p = 0.006). Conclusions: vitD₃ repletion status is an independent predictor of MMP-10 levels in T2D patients. Perhaps, high 25(OH)D₃ values should be targeted in these patients in order to prevent vascular complications.     

Induction of neutralizing antibodies by poliovirus capsid polypeptides VP1, VP2 and VP3

The antigenic and immunogenic properties of poliovirus capsid polypeptides (VPs) have been investigated in vivo and in vitro. VPs were isolated from infectious and formalin inactivated virus. Upon immunization VPs from infectious virus did not induce neutralizing antibodies in laboratory animals after two or three injections. Monomeric VP1, VP2 and VP3 from formalin inactivated virus all induced high titres of neutralizing antibodies generally already after two injections. Some anti-VP sera neutralized heterologous virus types. All anti-VP sera reacted with the corresponding capsid polypeptides from the homologous and heterologous virus strains in an immunoblotting assay. The anti-VP sera reacted equally well with D- and C-antigen in ELISA. Our results indicate that not only VP1, but also VP2 and VP3 are involved in the induction of neutralizing antibodies.     

Correlation between 25-hydroxyvitamin D/D3 Deficiency and COVID-19 Disease Severity in Adults from Northern Colorado

Vitamin D deficiency is common in the United States and leads to altered immune function, including T cell and macrophage activity that may impact responses to SARS-CoV-2 infection. This study investigated 131 adults with a history of a positive SARS-CoV-2 nasopharyngeal PCR and 18 adults with no COVID-19 diagnosis that were recruited from the community or hospital into the Northern Colorado Coronavirus Biorepository (NoCo-COBIO). Participants consented to enrollment for a period of 6 months and provided biospecimens at multiple visits for longitudinal analysis. Plasma 25-hydroxyvitamin D levels were quantified by LC-MS/MS at the initial visit (n = 149) and after 4 months (n = 89). Adults were classified as deficient (<30 nM or <12 ng/mL), insufficient (<30–50 nM or 12–20 ng/mL), or optimal (50–75 nM or >20 ng/mL) for 25-hydroxyvitamin D status. Fisher’s exact test demonstrated an association between disease severity, gender, and body mass index (BMI) at baseline. Mixed model analyses with Tukey-Kramer were used for longitudinal analysis according to BMI. Sixty-nine percent (n = 103) of the entire cohort had optimal levels of total 25(OH)D, 22% (n = 32) had insufficient levels, and 9% (n = 14) had deficent levels. Participants with severe disease (n = 37) had significantly lower 25-hydroxyvitamin D (total 25(OH)D) when compared to adults with mild disease (p = 0.006) or no COVID-19 diagnosis (p = 0.007). There was 44% of the cohort with post-acute sequalae of COVID-19 (PASC) as defined by experiencing at least one of the following symptoms after 60 days’ post-infection: fatigue, dyspnea, joint pain, chest pain, forgetfulness or absent-mindedness, confusion, or difficulty breathing. While significant differences were detected in 25-hydroxyvitamin D status by sex and BMI, there were no correlations between 25-hydroxyvitamin D for those without and without PASC. This longitudinal study of COVID-19 survivors demonstrates an important association between sex, BMI, and disease severity for 25-hydroxyvitamin D deficiency during acute stages of infection, yet it is not clear whether supplementation efforts would influence long term outcomes such as developing PASC.     

海南汉族血清维生素D水平与骨质疏松相关性

目的探讨海南汉族血清维生素D水平与骨质疏松的关系。方法选择年龄50～60岁骨质疏松患者334例及相应年龄段正常对照301例进行骨密度、血清25羟维生素D3水平测定。结果维生素D水平在两组之间存在显著差异。结论海南汉族血清中维生素D3水平下降与骨质疏松症的发生密切相关,对海南人群骨质疏松症的预防及治疗具有指导意义。     

四川省0~3岁婴幼儿25-羟维生素D营养状况的调查

目的：了解儿童保健门诊0~3岁婴幼儿25-羟维生素D水平及缺乏率。方法对2013年4月至2014年3月在四川省妇幼保健院儿童保健门诊就诊的0~3岁儿童1941例进行维生素D检测。结果0~3岁婴幼儿维生素D平均水平为(34.2±16.9)ng/mL,不同性别差异无统计学意义(t=1.46,P=0.14)。0~3岁婴幼儿维生素D缺乏率分别为3.4%、3.1%、7.0%、18.8%。不同年龄组维生素D水平及维生素D缺乏率差异有统计学意义(t=3.95,χ2=8.1;均P<0.05)。结论研究对象中2~3岁幼儿25-羟维生素D水平相对较低,2岁后维生素D缺乏有逐年升高的趋势,值得关注。     

待产女性血清维生素B12和叶酸水平的流行病学特征

目的 分析陕西省待产女性维生素B₁₂和叶酸水平及两者间关系的流行病学特征。方法 资料来源于2014年1月至2016年12月在6所三甲医院待产的健康孕妇,产前采集血样以测定维生素B₁₂和叶酸水平。采用分位数回归模型分析待产女性维生素B₁₂和叶酸水平间的关系。结果 共纳入1 277名待产女性,其血清维生素B₁₂的中位数为164.7 pg/ml,缺乏率为69.6%,血清叶酸的中位数为7.6 ng/ml,缺乏率为12.1%,单纯缺乏维生素B₁₂者占58.4%,单纯缺乏叶酸者占0.9%。农村待产女性维生素B₁₂和叶酸水平低于城市女性,维生素B₁₂和叶酸水平随年龄的增长而呈不同幅度增加,<25岁者维生素B₁₂和叶酸水平明显较低。叶酸缺乏者的维生素B₁₂水平比非缺乏者平均低37.62 pg/ml,分位数回归模型显示,在不同的维生素B₁₂分位点上,叶酸缺乏者的维生素B₁₂水平明显低于不缺乏者,且这种差异随着维生素B₁₂水平的升高而增大。结论 陕西省待产女性维生素B₁₂和叶酸缺乏症仍普遍存在,在妇幼保健实践中,叶酸增补的同时可能需重视维生素B₁₂补充。加大健康教育力度,重点提高农村和年轻女性对营养素补充重要性的认识,从而改善其维生素B₁₂和叶酸水平。     

血清25羟维生素D3和C反应蛋白与冠心病的相关性

目的　探讨血清25羟维生素D3［25(OH)D3］和C反应蛋白(CRP)与冠心病的相关性。方法　选择本院心脏中心疑似冠心病行冠状动脉造影检查的住院患者416例,冠状动脉狭窄Gensini积分表示冠状动脉病变的严重程度,分别检测血清25(OH)D3和CRP。根据冠状动脉造影结果分为正常冠状动脉组246例、稳定型心绞痛组110例、急性冠状动脉综合征(ACS)组60例。结果　正常冠状动脉组、稳定型心绞痛组、ACS组血清25(OH)D3分别为(33.15±20.95)、(28.93±16.45)、(15.55±5.70)nmol/L,正常冠状动脉组、稳定型心绞痛组与ACS组比较,差异均有统计学意义(均P＜0.001);正常冠状动脉组、稳定型心绞痛组、ACS组血清CRP分别为(4.33±0.12)、(5.68±0.25)、(5.73±0.31)mg/L,ACS组、稳定型心绞痛组与正常冠状动脉组比较,差异均有统计学意义(均P＜0.001)。正常冠状动脉组25(OH)D3与CRP及Gensini评分未见相关性;ACS组25(OH)D3与CRP、Gensini评分呈负相关(r=-0.026,P=0.045;r=-0.256,P=0.048),CRP与Gensini评分呈正相关(r=0.459,P＜0.001);稳定型心绞痛组25(OH)D3与CRP、Gensini评分呈负相关(r=-0.211,P=0.027;r=-0.208,P=0.029),CRP与Gensini评分呈正相关(r=0.574,P＜0.001)。结论　低水平的25(OH)D3与冠状动脉狭窄病变相关,结合CRP,低25(OH)D3可能通过炎症机制参与了冠心病的发病过程。     

Ultra-Marathon-Induced Increase in Serum Levels of Vitamin D Metabolites: A Double-Blind Randomized Controlled Trial

Purpose: While an increasing number of studies demonstrate the importance of vitamin D for athletic performance, the effects of any type of exercise on vitamin D metabolism are poorly characterized. We aimed to identify the responses of some vitamin D metabolites to ultra-marathon runs. Methods: A repeated-measures design was implemented, in which 27 amateur runners were assigned into two groups: those who received a single dose of vitamin D₃ (150,000 IU) 24 h before the start of the marathon (n = 13) and those (n = 14) who received a placebo. Blood samples were collected 24 h before, immediately after, and 24 h after the run. Results: In both groups of runners, serum 25(OH)D₃, 24,25(OH)₂D₃, and 3-epi-25(OH)D₃ levels significantly increased by 83%, 63%, and 182% after the ultra-marathon, respectively. The increase was most pronounced in the vitamin D group. Body mass and fat mass significantly decreased after the run in both groups. Conclusions: Ultra-marathon induces the mobilization of vitamin D into the blood. Furthermore, the 24,25(OH)₂D₃ and 3-epi-25(OH)D₃ increases imply that the exercise stimulates vitamin D metabolism.     

Vitamin D Metabolite Ratio in Pregnant Women with Low Blood Vitamin D Concentrations Is Associated with Neonatal Anthropometric Data

Existing evidence on the correlation between maternal vitamin D concentrations and birth outcomes is conflicting. Investigation of these associations requires accurate assessment of vitamin D status, especially in individuals with low 25-hydroxyvitamin D (25(OH)D) concentrations. This study examined the correlations between birth outcomes and the maternal vitamin D metabolite ratio (VMR) 1 (defined as the ratio of 24,25(OH)₂D₃ to 25(OH)D) and VMR2 (defined as the ratio of 3-epi-25(OH)D₃ to 25(OH)D) using data from the Japan Environment and Children’s Study at Chiba Regional Center. A total of 297 mother–neonate pairs were analyzed. Using liquid chromatography–tandem mass spectrometry, we measured 25(OH)D₂, 25(OH)D₃, 24,25(OH)₂D₃, and 3-epi-25(OH)D₃ concentrations in maternal serum samples. These data were analyzed in relation to birth anthropometric data using multivariable linear regression. Of the study participants, 85.2% showed insufficient vitamin D concentrations. VMR1 was strongly correlated with 25(OH)D concentrations, whereas VMR2 showed a weak correlation. Only VMR2 was associated with all anthropometric data. VMR2 in pregnant women with low vitamin D blood concentrations is a useful marker for neonatal anthropometric data and is independent of 25(OH)D. Accurate measurement of vitamin D metabolites could help better understand the effects of vitamin D on birth outcomes.     

Factors Associated with Serum Vitamin D Metabolites and Vitamin D Metabolite Ratios in Premenopausal Women

The most representative indicator of vitamin D status in clinical practice is 25(OH)D₃, but new biomarkers could improve the assessment of vitamin D status and metabolism. The objective of this study is to investigate the association of serum vitamin D metabolites and vitamin D metabolite ratios (VMRs) with potentially influential factors in premenopausal women. This is a cross-sectional study based on 1422 women, aged 39–50, recruited from a Madrid Medical Diagnostic Center. Participants answered an epidemiological and a food frequency questionnaire. Serum vitamin D metabolites were determined using an SPE–LC–MS/MS platform. The association between participant’s characteristics, vitamin D metabolites, and VMRs was quantified by multiple linear regression models. Mean 25(OH)D₃ concentration was 49.2 + 18.9 nmol/L, with greater deficits among obese, nulliparous, dark-skinned women, and with less sun exposure. A lower R2 ratio (1,25(OH)₂D₃/25(OH)D₃) and a higher R4 (24,25(OH)₂D₃/1,25(OH)₂D₃) were observed in nulliparous women, with high sun exposure, and those with low caloric intake or high consumption of calcium, vitamin D supplements, or alcohol. Nulliparous women had lower R1 (25(OH)D₃/Vit D₃) and R3 (24,25(OH)₂D₃/25(OH)D₃), and older women showed lower R3 and R4. Vitamin D status modified the association of the VMRs with seasons. VMRs can be complementary indicators of vitamin D status and its endogenous metabolism, and reveal the influence of certain individual characteristics on the expression of hydroxylase enzymes.     

PRESENCE OF 1, 25-DIHYDROXYVITAMIN D3 RECEPTOR IN RAT PITUITARY

A cytosolic receptor, specific for 1,25-dihydroxyvitamin D₃ (DHD) is present in the pituitary gland of the rat. Its properties are similar to the DHD receptors found in other tissues.