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。     

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.     

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.     

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。     

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.     

Effects of sun exposure and dietary vitamin D intake on serum 25-hydroxyvitamin D status in hemodialysis patients

BACKGROUND/OBJECTIVES

Vitamin D deficiency is common in hemodialysis patients. The aim of this study was to identify whether or not sun exposure and dietary vitamin D intake have effects on serum 25-hydroxyvitamin D (25(OH)D) status in hemodialysis (HD) patients. The objective was to identify the main determinants of serum vitamin D status in the study subjects.

SUBJECTS/METHODS

A cross-sectional study of 47 HD patients (19 males and 28 females) was performed. We assessed serum 25(OH)D and 1,25(OH)₂D levels between August and September 2012 and analyzed the prevalence of vitamin D deficiency in HD patients. To evaluate the determinants of serum 25(OH)D levels, we surveyed dietary vitamin D intake, degree of sun exposure, and outdoor activities. To compare biological variables, serum 25(OH)D was stratified as below 15 ng/ml or above 15 ng/ml.

RESULTS

Mean 25(OH)D and 1,25(OH)₂D levels were 13.5 ± 5.8 ng/ml and 20.6 ± 11.8 pg/ml, respectively. The proportions of serum 25(OH)D deficiency (< 15 ng/ml), insufficiency (15-< 30 ng/ml), and sufficiency (≥ 30 ng/ml) in subjects were 72.4%, 23.4%, and 4.3%, respectively. Prevalence of vitamin D deficiency in female patients was 78.6%, whereas that in males was 63.2% (P = 0.046). Vitamin D intake and sun exposure time were not significantly different between the two stratified serum 25(OH)D levels. Dietary intake of vitamin D did not contribute to increased serum 25(OH)D levels in HD patients. The main effective factors affecting serum 25(OH)D status were found to be the sun exposure and active outdoor exercise.

CONCLUSIONS

Hypovitaminosis D is common in HD patients and is higher in females than in males. Sun exposure is the most important determinant of serum 25(OH)D status in HD patients.     

海口地区0~6岁儿童25羟维生素D水平及维生素D受体基因多态性与骨密度关联性分析

目的 调查海口地区0~6岁儿童血清25羟维生素D[25-（OH）D]水平及维生素D受体（VDR）基因多态性与骨密度（BMD）的关联性。方法 选取2020年1—12月在海南省某专科医院医学中心进行健康体检的0~6岁健康儿童作为调查对象,检测其血清25-（OH）D水平、VDR基因多态性及BMD,比较不同性别、年龄、体质指数（BMI）、BMD儿童的血清25-（OH）D水平,比较BMD正常和异常儿童的VDR基因多态性,分析VDR基因多态性与BMD的关联性。结果 共纳入1 580名0~6岁健康儿童,男童838人,女童742人,平均年龄（2.49±1.20）岁。血清25-（OH）D水平为（34.66±5.87）ng/mL,维生素D缺乏、不足、充足儿童比例分别为4.49%、21.01%、74.49%。不同性别儿童血清25-（OH）D水平、维生素D营养状态分布差异无统计学意义（均P>0.05）;不同年龄、BMI、BMD儿童血清25-（OH）D水平、维生素D营养状态分布差异有统计学意义（P<0.05或P<0.01）。基因分型检测结果显示,0~6岁健康儿童VDR基因ApaⅠ位点存在多态性,基因型为AA、Aa、aa。BMD正常和异常儿童VDR基因ApaⅠ位点基因型、等位基因分布差异有统计学意义（均P<0.01）。多因素Logistic回归分析结果显示,VDR基因ApaⅠ位点基因型aa型（OR=3.729）、携带a等位基因（OR=2.656）儿童发生BMD异常的风险较高。结论 海口地区0~6岁儿童血清25-（OH）D水平与儿童年龄、BMI、BMD有关,且儿童VDR基因多态性与BMD异常的发生密切相关。     

A crossover trial of high and low sucrose-carbohydrate diets in type II diabetics with hypertriglyceridemia.

Earlier work shows that hyperlipemic type II diabetics tolerate wide ranges of sucrose and carbohydrate intake without effects on glycemic control, but a rise of fasting serum triglycerides sometimes occurs. To address further the issue of individual susceptibility to carbohydrate, the current study was designed to use each patient as his own control when given diets widely varying in sucrose content. After a stabilization period in the hospital on a normal sucrose content diet, each subject was given either a very low sucrose (less than 3 gm/day)-low carbohydrate (38 +/? 2%) diet or a high sucrose (220 gm)-high carbohydrate (63 +/? 3%) diet for 4 weeks. On a separate admission the opposite diet was assessed, again after an initial normal sucrose content diet. No consistent differences occurred in serum glucose levels or in 24-hr urinary glycosuria. High sucrose-carbohydrate intake raised fasting hypertriglyceridemia after 2 weeks but less thereafter. Severe sucrose-carbohydrate restriction did not significantly decrease fasting serum triglycerides; postprandial triglycerides changed in a trend opposite to fasting levels. No differences occurred in fasting serum insulin or serum cholesterol levels, but postprandial insulin levels were higher in high sucrose-carbohydrate diets. A diet with low sucrose and low total carbohydrate appears to offer no improvement in glycemic control over at least 70-fold higher dietary sucrose levels. However, high sucrose and carbohydrate diets increase fasting triglyceride levels in hypertriglyceridemic type II diabetics.     

The proaggregatory effect of 25-hydroxycholecalciferol [25-(OH)D3] on human platelets

As a result of reported evidence for 1,25-dihydroxycholecalciferol [1-25-(OH)₂D₃] as a platelet agonist and in view of the fact that the metabolite 25-hydroxycholecalciferol [25-(OH)D₃] in human plasma greatly predominates in concentration (by approximately 1,000-fold) over 1,25-(OH)₂D₃, it was of considerable interest to investigate the potential for 25-(OH)D₃ to act as a proaggregating agent in human platelets. Platelet aggregation studies were conducted in an aggregometer using washed platelet suspensions to which was added vitamin D₃ or its metabolites. In contrast to the inactivity of vitamin D₃, the present results provide evidence for the capability of 25-(OH)D₃ to aggregate human platelets and relate its potential in this regard to other forms of vitamin D.