The anomalous behaviour of exogenous 25-hydroxyvitamin D in competitive binding assays

The Vitamin D International External Quality Assessment Scheme (DEQAS) was established in 1989 to monitor the performance of assays for 25-hydroxyvitamin D (25-OHD) and 1,25-dihydroxyvitamin D (I,25(OH)₂D). This is achieved through the quarterly distribution of five samples of human serum. Results are used to calculate an All-Laboratory Trimmed Mean and a Method Mean for each of the methods used by participants. In July 2005, participants were asked to assay serum to which 50.9 nmol of either 25-OHD₃ or 25-OHD₂ had been added as ethanolic solutions. The final concentration of ethanol in the serum was 0.7%. The distribution also included a sample of the original serum (OS) containing 0.7% pure ethanol. The percentage recoveries of exogenous 25-OHD₃ (R1) and 25-OHD₂ (R2) were calculated for each method. Results (OS nM, R1 and R2) were as follows: DiaSorin RIA (n = 53); 39.2, 82.1%, 83.3%, DiaSorin Liason (n = 16); 36.8, 81.4%, 88.6%, IDS RIA (n = 21); 36.4, 54.2%, 29.1%, IDS OCTEIA (n = 16); 47.3, 78.8%, 56.4%, Nichols Advantage (n = 21); 58.9, 46.4%, 43.2%, HPLC (n = 9); 42.6, 112.2%, 97.1%, LC–MS (n = 4); 34.0, 111.5%, 118.1%. The IDS RIA and Nichols assays gave unexpectedly low recoveries. This does not appear to be a calibration problem or the effect of ethanol.     

Circulating vitamin D3 and 25-hydroxyvitamin D in humans: An important tool to define adequate nutritional vitamin D status

Circulating 25-hydroxyvitamin D [25(OH)D] is generally considered the means by which we define nutritional vitamin D status. There is much debate, however, with respect to what a healthy minimum level of circulation 25(OH)D should be. Recent data using various biomarkers such as intact parathyroid hormone (PTH), intestinal calcium absorption, and skeletal density measurements suggest this minimum level to be 80 nmol (32 ng/mL). Surprisingly, the relationship between circulating vitamin D₃ and its metabolic product—25(OH)D₃ has not been studied. We investigated this relationship in two separate populations: the first, individuals from Hawaii who received significant sun exposure; the second, subjects from a lactation study who received up to 6400 IU vitamin D₃/day for 6 months.

Results (1) the relationship between circulating vitamin D₃ and 25(OH)D in both groups was not linear, but appeared saturable and controlled; (2) optimal nutritional vitamin D status appeared to occur when molar ratios of circulating vitamin D₃ and 25(OH)D exceeded 0.3; at this point, the V_max of the 25-hydroxylase appeared to be achieved. This was achieved when circulating 25(OH)D exceeded 100 nmol.

We hypothesize that as humans live today, the 25-hydroxylase operates well below its V_max because of chronic substrate deficiency, namely vitamin D₃. When humans are sun (or dietary) replete, the vitamin D endocrine system will function in a fashion as do these other steroid synthetic pathways, not limited by substrate. Thus, the relationship between circulating vitamin D and 25(OH)D may represent what “normal” vitamin D status should be.     

Circulating vitamin D3 and 25-hydroxyvitamin D in humans: An important tool to define adequate nutritional vitamin D status

Circulating 25-hydroxyvitamin D [25(OH)D] is generally considered the means by which we define nutritional vitamin D status. There is much debate, however, with respect to what a healthy minimum level of circulation 25(OH)D should be. Recent data using various biomarkers such as intact parathyroid hormone (PTH), intestinal calcium absorption, and skeletal density measurements suggest this minimum level to be 80 nmol (32 ng/mL). Surprisingly, the relationship between circulating vitamin D₃ and its metabolic product—25(OH)D₃ has not been studied. We investigated this relationship in two separate populations: the first, individuals from Hawaii who received significant sun exposure; the second, subjects from a lactation study who received up to 6400 IU vitamin D₃/day for 6 months.Results (1) the relationship between circulating vitamin D₃ and 25(OH)D in both groups was not linear, but appeared saturable and controlled; (2) optimal nutritional vitamin D status appeared to occur when molar ratios of circulating vitamin D₃ and 25(OH)D exceeded 0.3; at this point, the V_max of the 25-hydroxylase appeared to be achieved. This was achieved when circulating 25(OH)D exceeded 100 nmol.We hypothesize that as humans live today, the 25-hydroxylase operates well below its V_max because of chronic substrate deficiency, namely vitamin D₃. When humans are sun (or dietary) replete, the vitamin D endocrine system will function in a fashion as do these other steroid synthetic pathways, not limited by substrate. Thus, the relationship between circulating vitamin D and 25(OH)D may represent what “normal” vitamin D status should be.     

Serum 25-hydroxyvitamin D levels are not associated with impaired postural sway in community-dwelling older women: a 6-year follow-up study

Objectives:A positive association between levels of blood 25-hydroxyvitamin D (25[OH]D), an index of vitamin D status, and physical balance has been reported from cross-sectional studies, but longitudinal studies are rare. The present study aimed to test the hypothesis that low serum 25(OH)D levels are longitudinally associated with impaired postural sway over a 6-year follow-up period in older women.Methods:The present cohort consisted of 392 community-dwelling Japanese women aged ≥69 years. Baseline examinations included serum 25(OH)D and physical performance tests, including postural sway velocity. Standing postural sway was evaluated by measuring gravity-center sway velocity. Follow-up physical performance tests were conducted 6 years later.Results:Mean subject age and serum 25(OH)D levels were 73.3 years (SD 3.7) and 61.0 nmol/L (SD 16.9), respectively. No significant association was found between 25(OH)D levels and changes in postural sway velocity (adjusted P for trend=0.72). Women with 25(OH)D <30 nmol/L tended to have lower Δpostural sway velocity than those with 25(OH)D ≥30 nmol/L (mean, -0.59 vs 0.37 cm/s, respectively; adjusted P=0.13).Conclusions:Vitamin D levels are not longitudinally associated with impaired postural sway in older women. Further longitudinal studies are needed to corroborate the results of this study.     

Summer/winter differences in the serum 25-hydroxyvitamin D3 and parathyroid hormone levels of Japanese women

Serum 25-hydroxyvitamin D₃ [25(OH)D₃] is produced in the skin in response to exposure to ultraviolet radiation, and is a good indicator of vitamin D nutritional status. The aim of this study was to determine summer/winter differences in serum 25(OH)D₃ and parathyroid hormone (PTH) in Japanese women and how the summer and winter values are related. The subjects were 122 healthy Japanese women aged 45–81 years (average age: 65.7 years). They were medically examined twice, in September 1997 and February 1999. Serum 25(OH)D₃ and intact PTH were determined by high-performance liquid chromatography and a two-site immunoradiometric assay respectively. Lifestyle information was obtained through an interview. The seasonal differences (winter minus summer) in 25(OH)D₃ [Δ25(OH)D₃] and intact PTH concentrations were –18.8 nmol/l (SD 19.2, P<0.0001) and 0.98pmol/l (SD 1.02, P<0.0001) respectively. The correlation coefficient between summer (x) and winter (y) 25(OH)D₃ levels was 0.462 (P<0.0001), with a linearly fitted line of y=0.42x+26.4. This relationship was interpreted as subjects with higher summer 25(OH)D₃ values having greater reductions in winter 25(OH)D₃ concentrations. There were inter-individual differences in Δ25(OH)D₃, although the summer and winter 25(OH)D₃ concentrations were well-correlated. Since Δ25(OH)D₃ was not associated with any of the lifestyle factors, seasonal differences in the 25(OH)D₃ concentrations of an individual appeared to reflect her ability to produce 25(OH)D₃ photochemically in the skin. Sun bathing would be a less effective means of attaining adequate vitamin D nutritional status in a person with a small seasonal difference in 25(OH)D₃, i.e., one with a low 25(OH)D₃ level. Received: 17 December 1999 / Revised: 24 April 2000 / Accepted: 10 May 2000     

The assessment of circulating 25(OH)D and 1,25(OH)2D: where we are and where we are going

The field of Vitamin D assay technology has progressed significantly over the past 4 decades. Further, the clinical utility of these measurements has moved from esoteric into mainstream clinical diagnosis. This movement has been fueled by the realization that Vitamin D is involved in bodily systems beyond skeletal integrity. The clinical assay techniques for circulating 25(OH)D and 1,25(OH)₂D have progressed away from competitive protein binding assay (CPBAs) that utilize tritium reporters to radioimmunoassay (RIAs) that utilize both I¹²⁵ and chemiluminescent reporters. These advances have allowed direct serum analysis of 25(OH)D in an automated format that provides a huge sample throughput. Detection of circulating 25(OH)D can also be achieved utilizing direct high-performance liquid chromatographic (HPLC) or liquid chromatography coupled with mass spectrometry (LC–MS) techniques. These methods are accurate, however, they require expensive equipment and restrict sample throughput in the large clinical laboratory. Direct serum detection of 1,25(OH)₂D is unlikely to occur for many reasons as a sample pre-purification will always be required. However, a semi-automated chemiluminescent detection system with automated sample preparation is in final development for the determination of circulating 1,25(OH)₂D. These advances will allow both 25(OH)D and 1,25(OH)₂D to be detected in an accurate, rapid fashion to meet the clinical demands we see emerging.     

Twice single doses of 100,000 IU of vitamin D in winter is adequate and safe for prevention of vitamin D deficiency in healthy children from Ushuaia, Tierra Del Fuego, Argentina

In order to improve vitamin D status of children from Ushuaia (55°S), at the South of Argentina, double supplementation with 100.000 IU of vitamin D was administered at the beginning of winter (March 2004), and 3 months later during winter (June 2004). In 2004, serum 25-hydroxyvitamin D (25OHD) was measured before the first supplementation, a month after, and 3 months after receiving the second supplementation (March, April and September). We studied 18 healthy children from Ushuaia, age (mean ± S.D.) 7.3 ± 4.4 years old (range 1.2–14.6), seven girls and 11 boys. Before treatment, serum 25OHD was 29.3 ± 5.9 ng/ml. It increased significantly 1 month after the first supplementation (April): 35.3 ± 4.4 ng/ml (p < 0.001), and decreased significantly 3 months after the second supplementation: 22.4 ± 4.6 ng/ml (September (p < 0.001). No child was neither deficient (<10 ng/ml) nor insufficient (10–15 ng/ml) of vitamin D. On April, a month after the first supplementation, no children had vitamin D intoxication levels (>50 ng/ml). These results disclosed that to prevent vitamin D deficiency for children at zones of risk at the south of our country, double supplementation of 100,000 IU of vitamin D during autumn and winter, would be adequate and safe.     

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-M)⁻ and (M+NH₄)⁺ ions were monitored in the selected-ion monitoring mode.     

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.     

Real-life use of vitamin D3-fortified bread and milk during a winter season: the effects of CYP2R1 and GC genes on 25-hydroxyvitamin D concentrations in Danish families,the VitmaD study

Common genetic variants rs10741657 and rs10766197 in CYP2R1 and rs4588 and rs842999 in GC and a combined genetic risk score (GRS) of these four variants influence late summer 25-hydroxyvitamin D (25(OH)D) concentrations. The objectives were to identify those who are most at risk of developing low vitamin D status during winter and to assess whether vitamin D₃-fortified bread and milk will increase 25(OH)D concentrations in those with genetically determined low 25(OH)D concentrations at late summer. We used data from the VitmaD study. Participants were allocated to either vitamin D₃-fortified bread and milk or non-fortified bread and milk during winter. In the fortification group, CYP2R1 (rs10741657) and GC (rs4588 and rs842999) were statistically significantly associated with winter 25(OH)D concentrations and CYP2R1 (rs10766197) was borderline significant. There was a negative linear trend between 25(OH)D concentrations and carriage of 0–8 risk alleles (p < 0.0001). No association was found for the control group (p = 0.1428). There was a significant positive linear relationship between different quintiles of total vitamin D intake and the increase in 25(OH)D concentrations among carriers of 0–2 (p = 0.0012), 3 (p = 0.0001), 4 (p = 0.0118) or 5 (p = 0.0029) risk alleles, but not among carriers of 6–8 risk alleles (p = 0.1051). Carriers of a high GRS were more prone to be vitamin D deficient compared to carriers of a low GRS. Furthermore, rs4588-AA carriers have a low but very stable 25(OH)D concentration, and interestingly, also low PTH level.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-014-0413-7) contains supplementary material, which is available to authorized users.     

The assessment of circulating 25(OH)D and 1,25(OH)2D: Where we are and where we are going

The field of Vitamin D assay technology has progressed significantly over the past 4 decades. Further, the clinical utility of these measurements has moved from esoteric into mainstream clinical diagnosis. This movement has been fueled by the realization that Vitamin D is involved in bodily systems beyond skeletal integrity. The clinical assay techniques for circulating 25(OH)D and 1,25(OH)₂D have progressed away from competitive protein binding assay (CPBAs) that utilize tritium reporters to radioimmunoassay (RIAs) that utilize both I¹²⁵ and chemiluminescent reporters. These advances have allowed direct serum analysis of 25(OH)D in an automated format that provides a huge sample throughput. Detection of circulating 25(OH)D can also be achieved utilizing direct high-performance liquid chromatographic (HPLC) or liquid chromatography coupled with mass spectrometry (LC–MS) techniques. These methods are accurate, however, they require expensive equipment and restrict sample throughput in the large clinical laboratory. Direct serum detection of 1,25(OH)₂D is unlikely to occur for many reasons as a sample pre-purification will always be required. However, a semi-automated chemiluminescent detection system with automated sample preparation is in final development for the determination of circulating 1,25(OH)₂D. These advances will allow both 25(OH)D and 1,25(OH)₂D to be detected in an accurate, rapid fashion to meet the clinical demands we see emerging.     

25-hydroxyvitamin D (25-OHD) levels in Turkish geriatric population: A nationwide study

BackgroundAcross the world, 25-hydroxyvitamin D (25-OHD) deficiency is a major health problem associated with many chronic diseases in the geriatric population. Prior to this study, there were no data regarding 25-OHD levels among individuals over the age of 65 in Turkey. The aim of this study was to assess 25-OHD levels and seasonal variations in these values among people over the age of 65 in Turkey.MethodsThis study included vitamin D measurements taken in 2016, 2017, and 2018 from the Turkish population over the age of 65. The age, gender, and seasonal average data of the study population were defined. The study data were obtained from the database of the Ministry of Health, and a Kolmogorov-Smirnov test was used to assess the distribution of the data. Medians and interquartile ranges (IQRs) were calculated for all categories, as the data were nonparametric.ResultsThe number of vitamin D measurements taken from the geriatric individuals included in this study was 305,329 for 2016, 576,452 for 2017, and 752,837 for 2018. The medians and IQRs of the 25-OHD levels in this population were 16 μg/L (IQR 7.45-24.55 μg/L) for 2016, 16.1 μg/L (IQR 7.8-24.4 μg/L) for 2017, and 16.4 μg/L (IQR 8.95-23.85 μg/L) for 2018.ConclusionsWhile the 25-OHD levels of older men tended to increase during the period of seasonal sunlight in Turkey, this variability was observed in elderly women. This suggests that older women tend to live more sedentary lives and have insufficient sun exposure. Overall, the median 25-OHD levels of individuals over the age of 65 tended to decrease each year.     

Supplemental vitamin D increases serum cytokines in those with initially low 25-hydroxyvitamin D: A randomized,double blind,placebo-controlled study

The purpose of this study was to determine if vitamin D status before supplementation influences the cytokine response after supplemental vitamin D. Forty-six reportedly healthy adults (mean(SD); age, 32(7) y; body mass index (BMI), 25.3(4.5) kg/m²; serum 25-hydroxyvitamin D (25(OH)D), 34.8(12.2) ng/mL) were randomly assigned (double blind) to one of three groups: (1) placebo (n = 15), or supplemental vitamin D (cholecalciferol) at (2) 4000 (n = 14) or (3) 8000 IU (n = 17). Supplements were taken daily for 35 days. Fasting blood samples were obtained before (Baseline, Bsl) and 35-days after (35-d) supplementation. Serum 25(OH)D, 1,25-dihydroxyvitamin D (1,25(OH)D), cytokines, and intact parathyroid hormone with calcium were measured in each blood sample. Supplemental vitamin D increased serum 25(OH)D (4000 IU, ≈29%; 8000 IU, ≈57%) and 1,25(OH)D (4000 IU, ≈12%; 8000 IU, ≈38%) without altering intact parathyroid hormone or calcium. The vitamin D metabolite increases in the supplemental vitamin D groups (n = 31) were dependent on initial levels as serum 25(OH)D (r = −0.63, p < 0.05) and 1,25(OH)D (r = −0.45, p < 0.05) at Bsl correlated with their increases after supplementation. Supplemental vitamin D increased interferon (IFN)-γ and interleukin (IL)-10 in subjects that were vitamin D insufficient (serum 25(OH)D < 29 ng/mL) compared to sufficient (serum 25(OH)D ⩾ 30 ng/mL) at Bsl. We conclude that supplemental vitamin D increase a pro- and anti-inflammatory cytokine in those with initially low serum 25(OH)D.     

Review on the analysis of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and its phase I and phase II metabolites in biological matrices, foodstuff and beverages

The heterocyclic aromatic amine, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), has been shown to be carcinogenic in rodents, mice and rats. Following phase I N-hydroxylation and phase II esterification PhIP exerts its carcinogenic effect by binding to DNA purines. Quantitative and qualitative analysis of its bioactivated metabolites as well as it detoxification products is important in studying its biological effects and inter- and intra-individual exposures. A review is presented with an extensive coverage of publications specifically reporting on the analysis of PhIP and its phase I and II metabolites in biological matrices, foodstuff and beverages. Analytical techniques such as liquid and gas chromatography coupled with various detection techniques (mass spectrometry, ultraviolet or fluorescence detection) were mostly applied. We conclude that since the initial identification of PhIP in 1986 a large set of assays has been developed for the analysis of PhIP and its phase I and phase II metabolites in a wide range of matrices, these included food products and biological samples such as plasma, urine and faeces. In addition, it was shown that numerous metabolites were recovered and identified. Thus, we conclude that liquid chromatography coupled to mass spectrometry is clearly the method of choice for sensitive qualitative as well as quantitative analysis with high selectivity and reaching lower quantification levels in the sub pg/mL range. The main aim of this review is that it can be used by other researchers as a resource for method development and optimization of analytical methods of PhIP and its carcinogenic or detoxification products.     

Development of free 25-hydroxyvitamin D3 assay method using liquid chromatography-tandem mass spectrometry

The free hormone hypothesis has triggered controversies regarding the measurement of free vitamin D metabolites, such as free 25-hydroxyvitamin D (25(OH)D), as a suitable indicator for total vitamin D for clinical use. This issue can be addressed by developing a precise and accurate method for free 25(OH)D measurement. In the present study, a novel assay method for free 25(OH)D₃ based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed. Sample preparation first involved ultrafiltration to remove vitamin D-binding protein-bound and albumin-bound 25(OH)D, followed by extraction with a column, derivatization, evaporation, dissolution, and injection into the LC-MS/MS system. The coefficient of variation of repeatability and reproducibility obtained were 3.8–4.5% and 4.8–5.9%, respectively. Satisfactory linearity (r=0.999) was obtained up to 80 pg/ml. The lower quantification limit was 0.97 pg/ml and the S/N ratio on the peak of 1.0 pg/ml sample was 24.8 (which is more than the acceptable value of 10). The recovery rate was between 84.5 and 92.4% with a negligible matrix effect (94.5–104.9%). Levels of free 25(OH)D₃, but not total 25(OH)D₃, in the serum of the patients with chronic kidney disease (CKD) and hepatic cirrhosis (HC) were substantially lower than those in healthy subjects. The correlation coefficient between total and free 25(OH)D₃ was 0.738 in all samples, while the linear regression equations were different between the patients with CKD and HC. In conclusion, LC-MS/MS assay for free 25(OH)D₃ might be useful to evaluate high-throughput methods, including ELISA.     

Stereochemistry of naturally-occurring 25-hydroxyvitamin D3-26,23 lactone as determined by radioligand binding analysis and high-performance liquid chromatography

The four stereoisomers of 25-hydroxyvitamin D₃-26,23 lactone (25-OHD₃-26,23 lactone) were tested against in vivo 25-OHD₃-26,23 lactone to determine their relative competition in the radioligand binding assays for 25-OHD₃ and 1,25-(OH)₂D₃. The 25R-OHD₃-26,23S lactone and in vivo 25-OHD₃-26,23 lactone behaved identically in the radioligand binding assay for 25-OHD₃ and were ～5-fold more potent than 25-OHD₃ at displacing 25-OH[³H]D₃. The 25S-OHD₃-26,23S lactone was the poorest competitor in this assay, requiring a 10-fold excess relative to 25-OHD₃ to displace 50% of the 25-OH[³H]D₃. The order of competition in the 25-OHD₃ radioligand binding assay was 25R-OHD₃-26,23S lactone = in vivo 25-OHD₃-26,23 lactone ? 25S-OHD₃-26,23R lactone > 25-OHD₃ ? 25R-OHD₃-26,23R lactone > 25S-OHD₃-26,23S lactone. The order of competition in the 1,25-(OH)₂D₃ cytosol receptor assay was essentially reversed from the competition in the 25-OHD₃ assay and was 25S-OHD₃-26,23S lactone > 25-OHD₃ ? 25S-OHD₃-26,23R lactone > 25R-OHD₃-26,23S lactone = in vivo 25-OHD₃-26,23 lactone. When tested in a high-performance liquid chromatographic system which separates all four stereoisomers, the in vivo 25-OHD₃-26,23 lactone comigrated with synthetic 25R-OHD₃-26,23S lactone. These data firmly establish that the naturally-occurring 25-OHD₃-26,23 lactone has the 25R, 23S stereochemistry. In addition, these data are the first to demonstrate that the four stereoisomers of 25-OHD₃-26,23 lactone have different affinities for the plasma vitamin D binding protein and the 1,25-(OH)₂D cytosol receptor.     

Vitamin D receptor gene polymorphisms (TaqI and ApaI) in relation to 25-hydroxyvitamin D levels and coronary artery disease incidence

Abstract

Context/objective: Previous studies have illustrated the association of the ApaI and TaqI polymorphisms of the vitamin D receptor gene, located in non-coding and coding regions, respectively, with diseases such as cancer and cardiovascular disease; however, investigating such association in Egyptian patients with coronary artery disease (CAD) has never been formerly attempted. Materials and methods: Male patients (n?=?137), 35–50 years of age, with verified CAD, were recruited alongside age- and sex-matched controls (n?=?58). Genotyping and 25-hydroxyvitamin D [25(OH)D] measurement were performed by polymerase chain reaction RFLP and HPLC, respectively. Results: Comparison of the genotypic distribution of both the TaqI and ApaI polymorphisms between patients and controls yielded insignificant results (p?=?0.55 and 0.7, respectively). Comparison of the allelic distribution of both polymorphisms also yielded insignificant results. The TaqI polymorphism was not found to predict 25(OH)D levels, whereas the wild-type genotype of the ApaI polymorphism was associated with greater levels of 25(OH)D (p?=?0.02), taking all subjects into consideration. Discussion/conclusion: This study presents the ApaI and TaqI polymorphisms as non-influencing players in the pathogenesis of CAD in Egyptian males and the ability of only the ApaI polymorphism to predict 25(OH)D levels, thus warranting further investigations of the triangular relationship between the polymorphisms, 25(OH)D and CAD incidence.     

血清25-羟维生素D水平与儿童骨密度的相关性研究

目的:研究血清25-羟维生素D[25-(OH)D]水平与儿童骨密度(BMD)的相关性。方法:选择2017年1月到2017年12月在亳州市人民医院接受健康体检的儿童100例作为研究对象。根据血清25-(OH)D水平对维生素D(Vit D)营养状况进行分组,其中严重缺乏组9例,缺乏组28例,不足组42例和充足组21例。对比不同年龄段和不同性别儿童血清25-(OH)D、BMD水平以及不同Vit D营养状况儿童对应的BMD水平,并采用Spearman相关性分析法分析血清25-(OH)D水平与儿童BMD、年龄的相关性。结果:5-9岁和10-14岁儿童的血清25-(OH)D及BMD水平均分别低于1-4岁儿童,而10-14岁儿童又低于5-9岁儿童(P0.05)。男童的血清25-(OH)D及BMD水平均分别高于女童,差异有统计学意义(P0.05)。不足组、缺乏组、严重缺乏组儿童的BMD水平均分别低于充足组,且缺乏组和严重缺乏组低于不足组,严重缺乏组又低于缺乏组(P0.05)。根据Spearman相关性分析结果显示,血清25-(OH)D水平与儿童BMD呈正相关,而与年龄呈负相关(P0.05),年龄与儿童BMD呈负相关(P0.05)。结论:血清25-(OH)D水平与儿童BMD呈正相关,但与年龄则呈负相关,及时补充适量的Vit D以满足儿童的机体所需,有利于儿童健康成长。     

Frequency of fokI and taqI polymorphism of vitamin D receptor gene in Indian population and its association with 25-hydroxyvitamin D levels

BACKGROUND:

The VDR protein is at the centre of the vitamin D endocrine system, a complex physiological system with substantial feedback regulatory mechanisms involved in maintaining serum calcium and 1, 25 dihydroxy vitamin D3. Variations in VDR gene are shown to have implications in several diseases and have also been implicated as an important genetic factor affecting bone mass.

AIM:

To determine the frequency of Fok I and Taq I variants in healthy Indian individuals and its association with 25-OH-Vitamin D levels.

SETTINGS AND DESIGN:

Blood samples were collected from 143 unrelated normal individuals (Male-84 and Female-59) and their genotypes determined.

MATERIALS AND METHODS:

After amplification by polymerase chain reaction, each polymorphism was genotyped by restriction fragment length polymorphism. For 100 normal healthy individuals 25-hydroxyvitamin D estimation was done using DiaSorin kit method.

STATISTICAL ANALYSIS:

Graph pad software was used to calculate the P values from the Chi-square.

RESULTS:

Out of 143 samples analyzed for FokI and TaqI polymorphisms the following genotypic frequency was obtained FF 59%, Ff 36%, ff 5% and TT 49%, Tt 43%, tt 8% respectively.

CONCLUSIONS:

Results indicate that the distribution of the polymorphic loci Fok I and Taq I vary considerably not only in different populations, but also within India. Furthermore, when the genotypes were analyzed with respect to 25-OH-Vitamin D levels, a significant association was seen for the Taq 1 SNP but not with the Fok I.     

Effects of dietary 25-hydroxyvitamin D3 supplementation on growth performance,immune function and antioxidative capacity in weaned piglets

The study evaluated the effects of different doses of 25-hydroxyvitamin D₃ (25(OH)D₃) on growth performance, immune function and antioxidative capacity in piglets. In a 21-d trial, 35 weaned pigs were divided into five groups and diets were supplemented with 5.5 (control), 43.0, 80.5, 118.0 and 155.5 μg 25(OH)D₃/kg, respectively. No treatment effects were observed for average daily gain, average daily feed intake and feed to gain ratio. Increasing dietary 25(OH)D₃ levels increased serum 25(OH)D₃ concentrations linearly (p < 0.01), decreased the frequency of CD3+CD4+ and CD3+CD8+ T cells (p < 0.01), and the serum level of complement component 3 (p < 0.05). Supplementation of 80.5 and 118.0 μg 25(OH)D₃/kg enhanced the activity of serum glutathione peroxidase (p < 0.05) and addition of 43.0 μg 25(OH)D₃/kg increased the malondialdehyde concentration (p < 0.05). Overall, feeding high-dose 25(OH)D₃ to weaned pigs partly improved immune functions and the antioxidative capacity.