Accurate quantification of 25-hydroxyvitamin D3 (25(OH)D3), the gold-standard biomarker for vitamin D nutritional assessment, still presents considerable analytical challenges. Although 25(OH)D3 exhibits intrinsic photosensitivity and thermal lability, these properties can be effectively managed via standardized light-shielding and inert-atmosphere operations. Key bottlenecks for its immunoassay lie in empirical-driven antibody screening lacking systematic theoretical guidance and poor discrimination of structurally similar vitamin D metabolites. To address these bottlenecks, we explored the molecular-recognition mechanism of high-affinity monoclonal antibody mAb-1F3 toward its hapten using molecular-docking and binding-pocket analysis. Four key amino-acid residues (PHE 52, PHE 91, TYR 32, ILE 100) within the antibody complementarity-determining region form a cooperative binding network for specific recognition of the steroid nucleus and C25-hydroxyl moiety of 25(OH)D3. π-alkyl interactions from PHE 52, PHE 91 and TYR 32 stabilize the hydrophobic steroid skeleton, while ILE 100 forms a conserved hydrogen bond (2.8 Å) to realize specific recognition of the C25-hydroxyl group. This structurally synergistic binding mechanism endows mAb-1F3 with high affinity (IC50 = 24.8 ng/mL) and favorable metabolite-discriminating capability. Guided by this mechanistic insight, recombinant mAb-1F3 was prepared and integrated into a rapid colloidal-gold lateral-flow immunoassay (LFIA). Standard matrix-specific sample extraction procedures were adopted to mitigate matrix interference induced by the high lipophilicity of 25(OH)D3 in complex samples. The LFIA completes the detection within 5 min and shows satisfactory linear consistency with LC-MS/MS in raw milk, dietary supplement, and human serum matrices. This work establishes a structure-driven strategy for rational antibody characterization and design, provides critical mechanistic support for conventional empirical antibody-screening workflows, and facilitates performance improvement of vitamin D immunoanalytical methods.

This study was conducted to evaluate the effect of 25-hydroxycholecalciferol (25-OHD3) supplementation replacing vitamin D3 (VD3) in late gestation and lactation diets on reproductive and litter performance, milk composition, immunoglobulin concentrations, and antioxidant capacity in sows and their progeny. A total of 30 sows in late gestation (d 105-108 of gestation) were allotted to 3 treatments in 10 replicates and fed experimental lactation diets until weaning. Treatments were: 1) VD: basal diet with 2,000 IU/kg VD3 supplementation; 2) MIX: basal diet with 1,000 IU/kg VD3 + 1,000 IU/kg 25-OHD3 supplementation; and 3) 25D: basal diet with 2,000 IU/kg 25-OHD3 supplementation. Blood, colostrum, and milk samples were collected at farrowing and d 14 of lactation, and data were analyzed using PROC MIXED of SAS. No significant differences were observed in reproductive and litter performance among dietary treatments. Sows fed MIX and 25D had greater sow plasma 25-OHD3 levels compared to sows fed VD at farrowing and d 14 of lactation (P < 0.05), while sows fed the 25D had greater plasma 25-OHD3 levels than sows fed MIX at d 14 of lactation. Piglets from the 25D sows tended to have greater plasma 25-OHD3 levels than those from VD sows at birth (P = 0.07) with piglets from MIX sows having intermediate values. Sows fed 25D had greater colostrum fat content than those fed VD (P < 0.05). Sows fed the 25D had greater superoxide dismutase activity in plasma at farrowing (P < 0.05) and in colostrum (P = 0.06) than those fed VD and MIX. Sows fed MIX or 25D had greater plasma total antioxidant capacity (T-AOC) at farrowing (P < 0.05) than those fed the VD while sows fed the 25D had greater plasma T-AOC than those fed VD or MIX at d 14 of lactation (P < 0.05). Piglets from the MIX sows tended to have lower plasma malondialdehyde (P = 0.09) and greater plasma T-AOC (P = 0.06) at birth than those from VD sows, whereas no differences were observed in plasma superoxide dismutase activity. Sows fed MIX had greater colostrum immunoglobulin G concentrations than those fed VD (P < 0.05), although there was no difference in plasma immunoglobulin G concentrations in piglets. In conclusion, dietary 25-OHD3 supplementation partially or fully replacing VD3 in sow diets from late gestation through lactation improved vitamin D status, colostrum fat contents, immunoglobulin G concentrations, and sow and piglet antioxidant capacity without affecting reproductive or litter performance.

Accurate analysis of vitamin D in human milk is essential for infant nutrition but remains analytically challenging due to low levels and a complex matrix. This study evaluates a practical multi-method strategy using LC-MS/MS, ELISA, and UPLC-PDA for vitamin D analysis in human milk. LC-MS/MS precisely quantified 25(OH)D2 and 25(OH)D3 with assay LOQ of 0.045 ng/mL and 0.100 ng/mL, respectively. ELISA showed a 11.58 ng/mL positive bias of vitamin D which limits use for accurate quantification. UPLC-PDA profiled vitamins A and E effectively but lacked sensitivity for vitamin D. Longitudinally, LC-MS/MS results show 25(OH)D3 and total 25(OH)D concentrations remained relatively stable, with medians ranging from 0.141 to 0.281 ng/mL and 0.263 to 0.315 ng/mL, respectively, across the study period, while 25(OH)D2 peaked at 8 weeks before declining. This study establishes LC-MS/MS as the preferred analytical strategy for quantifying 25(OH)D2 and 25(OH)D3 in human milk, providing critical data to assess infant nutritional intake and guide clinical interventions.

This study was conducted to determine whether early dietary supplementation with an intermediate vitamin D metabolite could improve metabolic efficiency and liver function in rainbow trout. Over a 20-week feeding trial from first feeding, rainbow trout were fed either a control diet containing vitamin D3 alone or a diet additionally supplemented with 80 µg/kg 25-OH-D3. Plasma analysis confirmed that 25-OH-D3 was efficiently absorbed, with detectable levels of 25-OH-D3 and its epimer, indicating enhanced vitamin D status. While growth was similar between the experimental groups, fish receiving 25-OH-D3 exhibited a significantly improved feed efficiency, indicating a better nutrient utilization. Whole-body composition analysis revealed higher protein and ash content and reduced lipid levels, consistent with transcriptomic evidence of modulated cholesterol and bile acid metabolism. Transcriptome profiling revealed 1301 differentially expressed genes in the liver, with strong modulation of pathways related to vitamin D metabolism, lipid handling, bile acid recycling, and ferroptosis. Additional signatures suggested improved nutrient partitioning and metabolic efficiency, potentially mediated by transcriptional and post-transcriptional mechanisms, including mRNA splicing. Consistent with its role on genome regulation via the vitamin D receptor, the data support the role of an active vitamin D system as a key potent modulator of efficient hepatic metabolism and function supporting improved feed utilization and long-term health outcomes in aquaculture.

The first application of broadband cavity enhanced absorption spectroscopy (BBCEAS) as a detection method for high performance liquid chromatography (HPLC) in the ultraviolet (UV) spectral range of 250-400 nm is reported. A custom-designed HPLC-BBCEAS system, incorporating a laser-driven light source (LDLS) and high reflectivity cavity mirrors (R ≥ 0.99), was employed for the separation and quantification of the polycyclic aromatic hydrocarbons (PAHs) anthracene and pyrene at near-UV wavelengths (300-400 nm). Minimum detectable absorption coefficients, αmin(t) of 9.8 × 10[-6] cm[-1] Hz[-]½ and 9.6 × 10[-6] cm[-1] Hz[-]½ were achieved for anthracene and pyrene at 338 nm and 333 nm, respectively, with 6.8 × 10[-6] cm[-1] Hz[-]½ observed for anthracene at 356 nm. These values correspond to 25-fold and 18-fold improvements in sensitivity compared to a conventional Perkin-Elmer HPLC-UV system. In the deep-UV region (250-300 nm), the separation and detection of vitamin D2 and D3 were demonstrated using both the LDLS and a 280 nm LED with R ∼ 0.96 cavity mirrors. LDLS-based measurements yielded αmin(t) values of 5.1 × 10[-4] cm[-1] Hz[-]½ and 4.0 × 10[-4] cm[-1] Hz[-]½ for D2 and D3, respectively, while the LED measurements exhibited enhanced sensitivity with values of 7.7 × 10[-5] cm[-1] Hz[-]½ and 5.8 × 10[-5] cm[-1] Hz[-]½. These findings demonstrate that HPLC-BBCEAS enables broadband, multiplexed, and highly sensitive detection of UV-absorbing analytes and extends the practical detection range of HPLC into the deep-UV region.

Uric acid metabolism is associated with the development of type 2 diabetes mellitus (T2DM), cardiometabolic, and cardiovascular diseases. Additionally, T2DM patients often exhibit mild cognitive impairment (MCI). However, the underlying mechanisms remain unclear. This study aims to identify and validate biomarkers associated with uric acid metabolism in T2DM and MCI, with the goal of discovering potential diagnostic and therapeutic targets to improve the quality of life for T2DM patients. Transcriptomic data for T2DM, MCI and uric acid metabolism-related genes were sourced from public databases. Biomarkers were screened using machine learning and validated for expression. Subsequent analyses included functional enrichment, immune infiltration, subcellular localization, and drug prediction. Three biomarkers-HP, ITGB3, and SELP-were identified. All showed significantly elevated expression in the T2DM group (p < 0.05). HP and ITGB3 were primarily enriched in ribosome-related pathways, primary immunodeficiency, and adherens junction processes. Immune infiltration analysis revealed that immature B cells and plasmacytoid dendritic cells were significantly enriched in T2DM. HP showed the strongest positive correlation with plasmacytoid dendritic cells (cor = 0.65, FDR <0.05), while ITGB3 exhibited the strongest positive correlation with immature B cells (cor = 0.76, FDR <0.05). Several potential therapeutic drugs were predicted, including calcifediol (score = -99.93) and meclofenamic acid (score = -99.89). This study identified three candidate biomarkers co-dysregulated across T2DM and MCI transcriptomes and associated with uric acid metabolism. Given the exploratory sample sizes, these findings are considered hypothesis-generating and require validation in larger independent cohorts.

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by memory loss and behavioral changes. A pivotal influence on AD pathology is the dysregulation of microglia in the brain. Despite promising findings in mouse models, there are limitations to the translatable biological information across species due to differences in the physiology, timeline of disease, and human heterogeneity. To address these interspecies discrepancies, we developed a novel implementation of the Translatable Components Regression (TransComp-R) framework, which integrated microglial single-nucleus transcriptomic data to identify biological pathways in mice AD models predictive of human AD. We compared model variations with sparse and traditional principal component analysis (PCA), finding that standard PCA encoded more interpretable mouse PCs compared to sPCA despite limited differences in technical performance. Mouse PCs significantly differentiated human AD from control microglial cells in the BA41/42, BA6/8, hippocampus and entorhinal cortex brain regions. However, these PCs had limited separation of human AD from control microglia in the prefrontal cortex. Additionally, we identified gene signatures from FDA-approved drugs that correlated with significant mouse component loadings, including valproic-acid and calcifediol. This computational framework may support the discovery of cross-species disease similarities, including the identification of candidate pharmacological solutions that may translate across species.

The objectives of this study were to explore the effects of 25-hydroxyvitamin D3 on (1) colostrum yield and compositions, 25-hydroxyvitamin D3, minerals, and health of periparturient cows and (2) 25-hydroxyvitamin D3 and minerals status, growth performance, and health of their calves. Forty multiparous dry cows were assigned to either vitamin D3 group or 25-hydroxyvitamin D3 group. After calving, all their calves received colostrum from their own dams; calves were respectively assigned to one of four treatments: (1) calves from D-VD group were fed vitamin D3; (2) calves from D-VD group were fed 25-hydroxyvitamin D3; (3) calves from D-25D group were fed vitamin D3; (4) calves from D-25D group were fed 25-hydroxyvitamin D3. Feeding 25-hydroxyvitamin D3 significantly increased (p < 0.05) 25-hydroxyvitamin D3, calcium and phosphorus in serum and colostrum, serum antioxidant capacity and immunoglobulin G (IgG) of periparturient cows, and significantly increased (p < 0.05) serum 25-hydroxyvitamin D3, calcium, and phosphorus in calves at birth. Feeding 25-hydroxyvitamin D3 calves improved (p < 0.05) weaning weight, skeletal growth, and average daily gain together with increased antioxidant capacity and IgG. In conclusion, supplementing 25-hydroxyvitamin D3 to periparturient cows and their calves improved colostrum and health of cows and growth and health of their calves.

Vitamin D3 is an essential fat-soluble vitamin for the human body. Its metabolite, 25-hydroxyvitamin D3 (25(OH)D3), serves as the primary biomarker to assess vitamin D levels. The monitoring of 25(OH)D3 concentration is crucial for human health assessment. While traditional detection methods offer high sensitivity and accuracy, they are operationally complex and costly. This review systematically summarizes the most recent progress in 25(OH)D3 detection technologies. Special attention is given to the recognition modes of 25(OH)D3 by antibodies, nucleic acids, and molecularly imprinted recognition elements. Subsequently, the design strategies of diverse types of biosensors, including fluorescent, colorimetric, and electrochemical biosensors, are analyzed. Moreover, the development of portable devices, smartphone software, and flexible wearable devices for detection applications is also examined. Biosensing detection platforms are compared from the perspectives of target recognition, signal conversion, signal output, and application scenarios. Additionally, the potential of biosensor detection platforms in clinical diagnosis, health management, and community health surveillance is further investigated. Finally, the future trends of intelligent, portable, accurate, and home-use 25(OH)D3 detection systems are delineated. This review offers a comprehensive reference for researchers developing next-generation 25(OH)D3 diagnostic sensors and provides insights for the early prevention and treatment of vitamin D deficiency-related diseases.

Acute myocardial infarction (AMI) remains a leading cause of cardiovascular morbidity and mortality worldwide. Emerging evidence highlights vitamin D as a critical determinant of cardiovascular health. The CYP2R1 gene encodes the key 25-hydroxylase enzyme responsible for converting vitamin D to its principal circulating metabolite, 25-hydroxyvitamin D. However, the influence of CYP2R1 polymorphisms on AMI susceptibility, particularly within South Asian populations, has not been well characterized. This study investigates the association of two CYP2R1 variants, rs2060793 and rs12794714, with AMI risk and their relationship with serum vitamin D levels in a Bangladeshi cohort. A total of 502 participants comprising 251 AMI patients and 251 age- and sex-matched controls were analyzed. Genomic DNA was extracted and genotyped using PCR-RFLP, while serum 25-hydroxyvitamin D3 levels were quantified by HPLC. AMI patients exhibited markedly lower vitamin D concentrations (23.92 ± 0.94 ng/mL) than controls (30.3 ± 0.86 ng/mL; p < 0.0001). Genotypic analysis revealed a significant association between rs2060793 and AMI risk: the TC (OR = 2.49, 95% CI: 1.34-4.63) and CC (OR = 2.59, 95% CI: 1.37-4.90) genotypes conferred increased susceptibility compared to the TT genotype (p = 0.0064). The dominant model (TC + CC vs. TT) further confirmed this relationship (OR = 2.53, 95% CI: 1.39-4.61, p = 0.0016). In contrast, rs12794714 showed no significant association with AMI in this population. Stratified analysis indicated that rs2060793 was significantly linked to AMI in males but not females, while both variants were associated with increased risk in individuals aged ≤60 years, but not in those >60 years. Bioinformatic and molecular docking analyses (RegulomeDB, JASPAR, HADDOCK 2.4, DNAproDB) further demonstrated potential regulatory effects of these variants on CYP2R1 function. Collectively, our findings reveal a novel association between CYP2R1 rs2060793 and vitamin D deficiency with AMI risk in the Bangladeshi population, underscoring the interplay of genetic and metabolic determinants in the molecular pathogenesis of AMI.