The differentiation and assay of vitamins D2 and D3 by gas-liquid chromatography.
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ABSTRACT:
SUBMITTER: Murray TK
PROVIDER: S-EPMC1264829 | biostudies-other | 1966 Jan
REPOSITORIES: biostudies-other
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Project description: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.
| S-EPMC9547169 | biostudies-literature
Project description:ObjectivesClinical evaluation of vitamin D status is conventionally performed by measuring serum levels of a single vitamin D metabolite, 25-hydroxyvitamin D predominantly by immunoassay methodology. However, this neglects the complex metabolic pathways involved in vitamin D bioactivity, including two canonical forms D3 and D2, bioactive 1,25-dihydroxy metabolites and inactive 24-hydroxy and other metabolites.MethodsLiquid chromatography-tandem mass spectrometry (LC-MS/MS) can measure multiple analytes in a sample during a single run with high sensitivity and reference level specificity. We therefore aimed to develop and validate a LC-MS/MS method to measure simultaneously 13 circulating vitamin D metabolites and apply it to 103 human serum samples.ResultsThe LC-MS/MS method using a Cookson-type derivatization reagent phenyl-1,2,4-triazoline-3,5-dione (PTAD) quantifies 13 vitamin D metabolites, including mono and dihydroxy-metabolites, as well as CYP11A1-derived D3 and D2 metabolites in a single run. The lower limit of quantitation was 12.5 pg/mL for 1,25(OH)2D3 with accuracy verified by analysis of National Institute of Standards and Technology (NIST) 972a standards. Quantification of seven metabolites (25(OH)D3, 25(OH)D2, 3-epi-25(OH)D3, 20(OH)D3, 24,25(OH)2D3, 1,25(OH)2D3 and 1,20S(OH)2D3) was consistently achieved in human serum samples.ConclusionsThis profiling method can provide new insight into circulating vitamin D metabolite pathways forming the basis for improved understanding of the role of vitamin D in health and disease.
| S-EPMC8419114 | biostudies-literature
Project description:IntroductionThe measurement of water-soluble vitamins is essential to diagnose and monitor various vitamin deficiencies. Establishing stability limits for these vitamins is crucial to ensure accurate laboratory testing. This study aimed to assess the stability of commonly measured water-soluble vitamins under different storage conditions to improve the accuracy of water-soluble vitamins measurement.MethodsThe stability of thiamine, riboflavin, nicotinamide, pantothenic acid, pyridoxic acid and pyridoxal, biotin, 5-methyltetrahydrofolic acid (5-MTHF), and ascorbic acid was measured using liquid chromatography-tandem mass spectrometry. We investigated some pre-analytical factors: the effect of different storage temperatures and times variation between serum and plasma samples, and the impact of ice bath on the sample before centrifugation. We evaluated stability based on differences from the baseline.ResultsThiamine, pyridoxal, and ascorbic acid in serum exhibited instability at room temperature and 2-8°C. Riboflavin and 5-MTHF in serum were only stable for up to 48 and 72 h at 2-8°C. However, when stored at -20°C, all water-soluble vitamins remained stable for up to 72 h, whereas at -80°C, stability was maintained for up to 7 days. All vitamins in whole blood, except nicotinamide, were stable for up to 2-4 h when stored in an ice bath.ConclusionsWater-soluble vitamins, such as thiamine, riboflavin, pyridoxal, and ascorbic acid, are unstable at room temperature and 2-8°C. All vitamins were stable for up to 7 days and stored at -80°C. The ice bath improved the stability of whole blood samples before centrifugation. Thus, laboratories should ensure appropriate storage conditions to maintain pre-analytical quality for vitamin measurements.IntroductionThe measurement of water-soluble vitamins is essential to diagnose and monitor various vitamin deficiencies. Establishing stability limits for these vitamins is crucial to ensure accurate laboratory testing. This study aimed to assess the stability of commonly measured water-soluble vitamins under different storage conditions to improve the accuracy of water-soluble vitamins measurement.MethodsThe stability of thiamine, riboflavin, nicotinamide, pantothenic acid, pyridoxic acid and pyridoxal, biotin, 5-methyltetrahydrofolic acid (5-MTHF), and ascorbic acid was measured using liquid chromatography-tandem mass spectrometry. We investigated some pre-analytical factors: the effect of different storage temperatures and times variation between serum and plasma samples, and the impact of ice bath on the sample before centrifugation. We evaluated stability based on differences from the baseline.ResultsThiamine, pyridoxal, and ascorbic acid in serum exhibited instability at room temperature and 2-8°C. Riboflavin and 5-MTHF in serum were only stable for up to 48 and 72 h at 2-8°C. However, when stored at -20°C, all water-soluble vitamins remained stable for up to 72 h, whereas at -80°C, stability was maintained for up to 7 days. All vitamins in whole blood, except nicotinamide, were stable for up to 2-4 h when stored in an ice bath.ConclusionsWater-soluble vitamins, such as thiamine, riboflavin, pyridoxal, and ascorbic acid, are unstable at room temperature and 2-8°C. All vitamins were stable for up to 7 days and stored at -80°C. The ice bath improved the stability of whole blood samples before centrifugation. Thus, laboratories should ensure appropriate storage conditions to maintain pre-analytical quality for vitamin measurements.
| S-EPMC11797916 | biostudies-literature
Project description:Candida dubliniensis is often found in mixed culture with C. albicans, but its recognition is hampered as the color of its colonies in primary culture on CHROMagar Candida varies. Furthermore, definite identification of C. dubliniensis is difficult to achieve, time-consuming, and expensive. Therefore, a method to discriminate between these two closely related yeast species by fatty acid methyl ester (FAME) analysis using gas-liquid chromatography (Sherlock Microbial Identification System [MIS]; MIDI, Inc., Newark, Del.) was developed. Although the chromatograms of these two species revealed no obvious differences when applying FAME analysis, a new library (CADLIB) was successfully created using Sherlock Library Generation Software (MIDI). The amount and frequency of FAME was analyzed using library training files (n = 10 for each species), preferentially those comprising reference strains. For testing the performance of the CADLIB, clinical isolates genetically assigned to the respective species (C. albicans, n = 32; C. dubliniensis, n = 28) were chromatographically analyzed. For each isolate tested, MIS computed a similarity index (SI) indicating a hierarchy of possible strain fits. When using the newly created library CADLIB, the SIs for C. albicans and C. dubliniensis ranged from 0.11 to 0.96 and 0.53 to 0. 93 (for all but one), respectively. Only three isolates of C. albicans (9.4%) were misidentified as C. dubliniensis, whereas all isolates of C. dubliniensis were correctly identified. Resulting differentiation accuracy was 90.6% for C. albicans and 100% for C. dubliniensis. Cluster analysis and principal component analysis of the resulting FAME profiles showed two clearly distinguishable clusters matching up with two assigned species for the strains tested. Thus, the created library proved to be well suited to discriminate between these two species.
| S-EPMC87459 | biostudies-literature
Project description:BackgroundVitamin D deficiency increases the risk of developing multiple sclerosis (MS) but it is unclear whether vitamin D supplementation improves the clinical course of MS, and there is uncertainty about the dose and form of vitamin D (D2 or D3) to be used. The mechanisms underlying the effects of vitamin D in MS are not clear. Vitamin D3 increases the rate of differentiation of primary oligodendrocyte precursor cells (OPCs), suggesting that it might help remyelination in addition to modulating the immune response. Here we analyzed the transcriptome of differentiating rat CG4 OPCs treated with vitamin D2 or with vitamin D3 at 24 h and 72 h following onset of differentiation.MethodsGene expression in differentiating CG4 cells in response to vitamin D2 or D3 was quantified using Agilent DNA microarrays (n = 4 replicates), and the transcriptome data were processed and analysed using the R software environment. Differential expression between the experimental conditions was determined using LIMMA, applying the Benjamini and Hochberg multiple testing correction to p-values, and significant genes were grouped into co-expression clusters by hierarchical clustering. The functional significance of gene groups was explored by pathway enrichment analysis using the clusterProfiler package.ResultsDifferentiation alone changed the expression of about 10% of the genes at 72 h compared to 24 h. Vitamin D2 and D3 exerted different effects on gene expression, with D3 influencing 1272 genes and D2 574 at 24 h. The expression of the vast majority of these genes was either not changed in differentiating cells not exposed to vitamin D or followed the same trajectory as the latter. D3-repressed genes were enriched for Gene Ontology (GO) categories including transcription factors and the Notch pathway, while D3-induced genes were enriched for the Ras pathway.ConclusionsThis study shows that vitamin D3, compared with D2, changes the expression of a larger number of genes in OLs. Identification of genes affected by D3 in OLs should help to identify mechanisms mediating its action in MS.
| S-EPMC7146914 | biostudies-literature
Project description:A convenient method using commercial aqueous concentrated HCl (conc. HCl; 35%, w/w) as an acid catalyst was developed for preparation of fatty acid methyl esters (FAMEs) from sterol esters, triacylglycerols, phospholipids, and FFAs for gas-liquid chromatography (GC). An 8% (w/v) solution of HCl in methanol/water (85:15, v/v) was prepared by diluting 9.7 ml of conc. HCl with 41.5 ml of methanol. Toluene (0.2 ml), methanol (1.5 ml), and the 8% HCl solution (0.3 ml) were added sequentially to the lipid sample. The final HCl concentration was 1.2% (w/v). This solution (2 ml) was incubated at 45 degrees C overnight or heated at 100 degrees C for 1-1.5 h. The amount of FFA formed in the presence of water derived from conc. HCl was estimated to be <1.4%. The yields of FAMEs were >96% for the above lipid classes and were the same as or better than those obtained by saponification/methylation or by acid-catalyzed methanolysis/methylation using commercial anhydrous HCl/methanol. The method developed here could be successfully applied to fatty acid analysis of various lipid samples, including fish oils, vegetable oils, and blood lipids by GC.
| S-EPMC2817593 | biostudies-literature
Project description:In this study, salting-out assisted liquid-liquid extraction (SALLE) as a simple and efficient extraction technique followed by high-performance liquid chromatography (HPLC) was employed for the determination of vitamin D3 in milk samples. The sample treatment is based on the use of water-miscible acetonitrile as the extractant and acetonitrile phase separation under high-salt conditions. Under the optimum conditions, acetonitrile and ammonium sulfate were used as the extraction solvent and salting-out agent, respectively. The vitamin D3 extract was separated using Hypersil ODS (250x i.d 4.6 mm, 5 µm) HPLC column that was coupled with diode array detector. Vitamin D2 was used as internal standard (IS) to offset any variations in chromatographic conditions. The vitamin D3 and the IS were eluted in 18 min. Good linearity (r 2 > 0.99) was obtained within the range of 25-600 ng g-1 with the limit of detection of 15 ng g-1 and limit of quantification of 25 ng g-1. The validated method was applied for the determination of vitamin D3 in milk samples. The recoveries for spiked samples were from 94.4 to 113.5%.
| S-EPMC6731698 | biostudies-literature
Project description:Intact methionine residues in proteins were rapidly and precisely determined by measuring methyl thiocyanate released during the reaction with CNBr and separated by g.l.c. Conditions for the reaction and for chromatography on columns of Porapak P-S are described. The recovery of methyl thiocyanate from several methionine derivatives and analogues were examined. Carbamoylmethionine was adopted as a stable primary standard and ethyl thiocyanate as internal standard. The measured methionine content of several isolated proteins was close to the theoretical value indicated by previous work and the results for these and a range of food proteins agreed well with results obtained by ion-exchange chromatography after performic acid oxidation. Since CNBr does not react with methionine sulphoxide and a preliminary hydrolysis is not required, the method discriminates between methionine and any methionine sulphoxide that may be present. It could be useful in studies on the nutritional availability of methionine in processed foods.
| S-EPMC1172884 | biostudies-other
Project description:Dopamine D2 and D3 autoreceptors are located on pre-synaptic terminals and are known to control the release and synthesis of dopamine. Dopamine D3 receptors have a fairly restricted pattern of expression in the mammalian brain. Their localization in the nucleus accumbens core and shell is of particular interest because of their association with the rewarding properties of drugs of abuse. Using background subtracted fast scan cyclic voltammetry, we investigated the effects of dopamine D2 and D3 agonists on electrically stimulated dopamine release and uptake rates in the mouse caudate-putamen and nucleus accumbens core and shell. The dopamine D2 agonists (-)-quinpirole hydrochloride and 5,6,7,8-Tetrahydro-6-(2-propen-1-yl)-4H-thiazolo[4,5-d]azepin-2-amine dihydrochloride (B-HT 920) had the same dopamine release inhibition effects on caudate-putamen and nucleus accumbens (core and shell) based on their EC(50) and efficacies. This suggests that the dopamine D2 autoreceptor functionality is comparable in all three striatal regions investigated. The dopamine D3 agonists (4aR,10bR)-3,4a,4,10b-Tetrahydro-4-propyl-2H,5H-[1]benzopyrano-[4,3-b]-1,4-oxazin-9-ol hydrochloride ((+)-PD 128907) and (+/-)-7-Hydroxy-2-dipropylaminotetralin hydrobromide (7-OH-DPAT) had a significantly greater effect on dopamine release inhibition in the nucleus accumbens shell than in caudate-putamen. This study confirms that, the dopamine D3 autoreceptor functionality is greater in the nucleus accumbens shell followed by the nucleus accumbens core, with the caudate-putamen having the least. Neither dopamine D2 nor D3 agonists affected the uptake rates in nucleus accumbens but concentrations greater than 0.3 muM lowered the uptake rate in caudate-putamen. To validate our method of evaluating dopamine D2 and D3 autoreceptors, sulpiride (D2 antagonist) and nafadotride (D3 antagonist) were used to reverse the effects of the dopamine agonists to approximately 100% of the pre-agonist dopamine release concentration. Finally, these results demonstrate a functional voltammetric assay that characterizes dopamine D2-like agonist as either D2- or D3-preferring agonists by taking advantage of the unique receptor density within the striatum.
| S-EPMC2887711 | biostudies-literature
Project description:We describe the changes in gene expression profile of differentiating rat CG4 oligodendrocyte progenitor cell lines treated with either vitamin D2 or D3. The treatment was initiated in cells pre-differentiated for 2 days (time 0) and the changes in gene expression were monitored at 24 h and 72 h post-treatment.
2020-07-26 | E-MTAB-8098 | biostudies-arrayexpress