Quantitative analysis of vitamin D and its main metabolites in human milk by supercritical fluid chromatography coupled to tandem mass spectrometry.

A novel method to quantitate vitamin D and its main metabolites (vitamin D3, vitamin D2, and their 25-hydroxy metabolites) in breast milk by supercritical fluid chromatography has been developed and fully validated. A small volume of sample (1 mL) is subjected to ethanolic protein precipitation and liquid-liquid extraction. Final extracts are derivatized with 4-phenyl-1,2,4-triazoline-3,5-dione and vitamin D derivatives analyzed by supercritical fluid chromatography hyphenated to tandem mass spectrometry with atmospheric pressure chemical ionization. Multiple reaction monitoring is used for quantitation. Separation conditions were optimized using a gradient of methanol-water-ammonium formate into carbon dioxide. Make-up solvent was methanol containing ammonium formate. The quantitation limit reached levels as low as 50 pmol/L, with intra- and inter-day relative standard deviations lower than 15% and 20% for all analytes. Accuracy was evaluated by spiking experiments and was well within acceptability ratios (± 15%). The method was then applied to a subset of commercially available human milk samples. The newly developed method provides opportunities to determine the nutritional status of mother-infant dyads from a non-invasive measure, or for interventional or observational studies building knowledge on the composition of human milk. Graphical abstract.

Gene expression changes by high-polyphenols cocoa powder intake: a randomized crossover clinical study.

PURPOSE: To assess the effect of the intake of a single dose of high-polyphenols cocoa on gene expression in peripheral mononuclear cells (PBMCs), and analyze conjugated (-)-epicatechin metabolites in plasma, which may be related with an antioxidant response in healthy human. METHODS: A randomized, controlled, double-blind, cross-over, clinical trial in healthy young adults who consumed a single dose of high-polyphenols cocoa powder and maltodextrins as control, with a one-week washout period. Analysis of circulating metabolites, plasma antioxidant capacity and gene expression changes in PBMCs were performed under fasting conditions and 2-h after treatment using microarray in a subsample. Pathway analysis was conducted using Ingenuity Pathway Analysis (IPA). RESULTS: Twenty healthy participants (9 F) were included in the study. A significant increase in circulating (-)-epicatechin metabolites was found after cocoa intake in all participants without related changes in antioxidant capacity of plasma. The metabolites profile slightly varied across subjects. Treatments triggered different transcriptional changes in PBMC. A group of 98 genes showed changes in expression after cocoa treatment, while only 18 were modified by control. Differentially expressed genes included inflammatory cytokines and other molecules involved in redox balance. Gene and network analysis after cocoa intake converged in functions annotated as decreased production of reactive oxygen species (p = 9.58E-04), decreased leukocyte activation (p = 4E-03) and calcium mobilization (p = 2.51E-05). CONCLUSIONS: No association was found between conjugated metabolites in plasma and antioxidant capacity. Changes in PBMCs gene expression suggest anti-inflammatory effects.

Double-balloon jejunal perfusion to compare absorption of vitamin E and vitamin E acetate in healthy volunteers under maldigestion conditions.

BACKGROUND/OBJECTIVES: The vitamin E derivative, α-tocopheryl acetate, is often included in formulations used in enteral nutrition. In this respect, we compared α-tocopherol and α-tocopheryl acetate absorption under 'maldigestion' conditions, such as occurring during enteral tube feeding, using differentially labeled RRR-[5,7-methyl-((2)H(6))]-α-tocopherol and RRR-[5-methyl-(2)H(3)]-α-tocopheryl acetate allowing direct comparison between free and esterified forms. SUBJECTS/METHODS: The two derivatives were given together in a single dose to six volunteers directly into the jejunum using a double-balloon perfusion system. Perfusion lasted for 1 h, and the collected blood and effluent samples were analyzed by liquid chromatography-mass spectrometry. RESULTS: In the isolated 20-cm length of exposed jejunum, on average ~ 6% of the two vitamin E forms were absorbed >1 h based on subtraction of effluent from influent. There was substantial difference in the absolute absorbed quantity between individuals, but no significant differences were observed in the absorption between the two labeled forms as assessed in the plasma. (2)H(3)-α-tocopherol was not present in the influent, but appeared in the effluent, indicating that the acetylated form of vitamin E is cleaved by brush border enzymes in the small intestine. CONCLUSIONS: This study shows that even in the absence of digestive enzymes and bile salts, the appropriately solubilized acetylated form of α-tocopherol exhibits the same bioavailability as free α-tocopherol. This suggests that both forms can be absorbed equally under maldigestion conditions such as present clinically during enteral tube feeding.