Determination of Bovine Lactoferrin in Powdered Infant Formula and Adult Nutritionals by Heparin Affinity Extraction and Reverse-Phase High-Performance Liquid Chromatography/Ultraviolet Detection (HPLC/UV): Single-Laboratory Validation, First Action 2021.10.

BACKGROUND: Infant formulas, and pediatric and adult nutritional products, are being fortified with bovine lactoferrin (bLF) due to its beneficial impacts on immune development and gut health. Lactoferrin supplementation into these products requires an analytical method to accurately quantify the concentrations of bLF to meet global regulatory and quality standards. OBJECTIVE: To develop and validate a lactoferrin method capable of meeting the AOAC INTERNATIONAL Standard Method Performance Requirements (SMPR®) 2020.005. METHODS: Powder formula samples are extracted using warm dibasic phosphate buffer, pH 8, then centrifuged at 4°C to remove insoluble proteins, fat, and other solids. The soluble fraction is further purified on a HiTrap heparin solid-phase extraction (SPE) column to isolate bLF from interferences. Samples are filtered, then analyzed by LC-UV using a protein BEH C4 analytical column and quantitated using an external calibrant. RESULTS: The LOQ (2 mg/100 g), repeatability (RSD: 2.0-4.8%), recovery (92.1-97.7%), and analytical range (4-193 mg/100 g) all meet the method requirements as stated in SMPR 2020.005 for lactoferrin. CONCLUSION: The reported single-laboratory validation (SLV) results demonstrate the ability of this lactoferrin method to meet or exceed the method performance requirements to measure soluble, intact, non-denatured bLF in infant and adult nutritional powder formulas. HIGHLIGHTS: The use of a heparin affinity column to isolate lactoferrin from bovine milk products combined with a selective analytical chromatographic column provides suitable analyte specificity without requiring proprietary equipment or reagents.

The human milk component myo-inositol promotes neuronal connectivity.

Effects of micronutrients on brain connectivity are incompletely understood. Analyzing human milk samples across global populations, we identified the carbocyclic sugar myo-inositol as a component that promotes brain development. We determined that it is most abundant in human milk during early lactation when neuronal connections rapidly form in the infant brain. Myo-inositol promoted synapse abundance in human excitatory neurons as well as cultured rat neurons and acted in a dose-dependent manner. Mechanistically, myo-inositol enhanced the ability of neurons to respond to transsynaptic interactions that induce synapses. Effects of myo-inositol in the developing brain were tested in mice, and its dietary supplementation enlarged excitatory postsynaptic sites in the maturing cortex. Utilizing an organotypic slice culture system, we additionally determined that myo-inositol is bioactive in mature brain tissue, and treatment of organotypic slices with this carbocyclic sugar increased the number and size of postsynaptic specializations and excitatory synapse density. This study advances our understanding of the impact of human milk on the infant brain and identifies myo-inositol as a breast milk component that promotes the formation of neuronal connections.

cis- versus trans-ceramides: effects of the double bond on conformation and H-bonding interactions.

Natural ceramides (Cers) possess a trans double bond between C4 and C5 of the sphingoid chain. This double bond is critical to their cell signaling properties. Both a change from trans to cis and the saturation of this site lead to changes in or loss of biological activity. To explore the conformational impact of the cis double bond, through-bond, and through-space interactions were investigated in hydrated Cers by multidimensional (1)H and (13)C NMR spectroscopy. Unlike trans-Cer, the cis-isomer exhibited not one but two broad yet resolved resonances for the protons in C1-OH and C3-OH, much like dihydroceramide (DHCer). Temperature-dependent studies and partial isotopic labeling of cis-Cer revealed that relative to trans-Cer, these two OH groups form weaker hydrogen bonds, particularly in the case of C1-OH. Our results also suggest that the cis double bond twists, slightly, the orientation of HO-C1 with respect to HO-C3, thus weakening the hydrogen-bonding network formed between the two OH groups of cis-Cer and bound water molecules. The alteration of the local network of H-bonds may account for the differences observed in the biological activity of the two isomers.