Protein-ligand docking using FFT based sampling: D3R case study.

Fast Fourier transform (FFT) based approaches have been successful in application to modeling of relatively rigid protein-protein complexes. Recently, we have been able to adapt the FFT methodology to treatment of flexible protein-peptide interactions. Here, we report our latest attempt to expand the capabilities of the FFT approach to treatment of flexible protein-ligand interactions in application to the D3R PL-2016-1 challenge. Based on the D3R assessment, our FFT approach in conjunction with Monte Carlo minimization off-grid refinement was among the top performing methods in the challenge. The potential advantage of our method is its ability to globally sample the protein-ligand interaction landscape, which will be explored in further applications.

Assessment of 3-epi-25-hydroxyvitamin D levels during cholecalciferol supplementation in adults with chronic liver diseases.

Recently, hepatic immaturity was cited as a possible reason for high levels of the C-3 epimer of 25-hydroxyvitamin (25(OH)D) in premature infants: however what role, if any, the liver plays in controlling epimer concentrations is unknown. This study assesses 3-epi-25-hydroxyvitamin D (3-epi-25(OH)D) levels during the course of cholecalciferol supplementation in adults with chronic liver diseases (CLD). Vitamin D metabolites were analyzed in 65 CLD patients with 25(OH)D <30 ng/mL who received 20 000 IU cholecalciferol/week for 6 months. The primary outcome assessed serum 25(OH)D and 3-epi-25(OH)D in response to supplementation. Corresponding values from 16 CLD patients with sufficient vitamin D levels receiving no supplementation were compared. The epimer was detected in all samples and at lower relative concentrations with lower vitamin D baseline status, i.e., severe vitamin D deficiency (<10 ng/mL) as compared with deficient (10-19.9 ng/mL), insufficient (20-29.9 ng/mL), or sufficient (≥30 ng/mL) vitamin D levels (2.4% vs. 4.8%, 5.2%, 5.8%, respectively; P < 0.001). Similar relative concentrations for 3-epi-25(OH)D, ranging from 4.3%-7.1% (absolute concentrations: 1.1-4.0 ng/mL; all P < 0.001), were obtained in response to cholecalciferol in all supplemented patients, regardless of inadequacy threshold. Epimer levels significantly decreased (P = 0.007) in unsupplemented patients, coinciding with decreasing serum 25(OH)D concentrations over time. No epimer differences between patients with (n = 17) or without (n = 48) cirrhosis were demonstrated. The 3-epi-25(OH)D was present in serum of all patients at comparable levels to those reported by others. Epimer levels increased linearly with increasing 25(OH)D levels after supplementation. However, no effect of cirrhosis on epimer concentrations was observed.

Full-scan mass spectral evidence for 3-epi-25-hydroxyvitamin D3 in serum of infants and adults.

BACKGROUND: Since the introduction of liquid chromatography-mass spectrometry (LC/MS) for assessing vitamin D status, it has been shown that the C-3 epimer can account for a significant proportion of circulating 25-hydroxyvitamin D3 (25OHD3) concentrations in infants. However, some question whether monitoring a single MS transition at a chromatographic retention time typical for 3-epi-25OHD3 sufficiently warrants conclusions about the identity of the substance generating the signal. Therefore, we aimed to substantiate the evidence for 3-epi-25OHD3 in infants by collision induced dissociation (CID)-MS/MS product ion scans. A second objective was mass spectrometric investigation of the presence and prevalence of the 3-epi metabolite in serum from adults. METHODS: Serum samples from six infants and 32 adults were studied using an ultra performance LC/tandem MS (UPLC/MS/MS) method designed to separate the 3-epi-25OHD3 from 25OHD3. Samples were submitted to liquid/liquid extraction and Sephadex LH-20 fractionation, prior to column-switching UPLC with MS/MS recording of CID product ion spectra of the [M+H](+) precursor ion. The respective standards were analyzed under identical UPLC/MS/MS conditions for comparison. RESULTS: In the chromatograms of all samples, two peaks eluted with retention characteristics and spectra closely matching those observed for the 25OHD3 and the 3-epi standards. The percentage of the 3-epi metabolite relative to 25OHD3 in infants ranged from 15% to 41%, and in adults from 2.5% to 17%. CONCLUSIONS: This preliminary finding suggests that the prevalence of 3-epi-25OHD3 in serum of infants is considerable, and that even in adults the concentrations of this form should not be neglected.

Liquid chromatography-tandem mass spectrometric method for the determination of salivary 25-hydroxyvitamin D3: a noninvasive tool for the assessment of vitamin D status.

A sensitive liquid chromatography-electrospray ionization-tandem mass spectrometric (LC-ESI-MS/MS) method for the determination of 25-hydroxyvitamin D(3) [25(OH)D(3)] in human saliva has been developed and validated. The saliva was deproteinized with acetonitrile, purified using a Strata-X cartridge, derivatized with a Cookson-type reagent, 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD), and subjected to LC-MS/MS. The PTAD derivative was much more easily ionized in positive-ESI-MS and efficiently produced a characteristic product ion during MS/MS, compared to the intact 25(OH)D(3). Methylamine was used as the mobile phase additive, and also effectively enhanced the assay sensitivity. Quantification was based on selected reaction monitoring, and 25-hydroxyvitamin D(4) was used as the internal standard. This method allowed the reproducible and accurate quantification of salivary 25(OH)D(3) using a 1.0-ml sample, and the limit of quantitation for 25(OH)D(3) was 2.0 pg/ml. The applicability of the developed method for clinical studies was then examined. There was a positive linear relationship (r (2) = 0.830) between the serum 25(OH)D(3) level, which is conventionally used as a means of assessing the vitamin D status, and the salivary 25(OH)D(3) level measured using the proposed method. The method also enabled the detection of the increase in the salivary 25(OH)D(3) level after the supplementation of vitamin D(3).