Development of a mass spectrometry diagnostic assay to monitor hepcidin levels in hemochromatosis patients

Hepcidin has been identified as the principle regulatory peptide essential for iron homeostasis. Expression of this cysteine-rich peptide is up-regulated by inflammation and iron overload, whereas hypoxia, iron deficiency and erythropoiesis suppress hepcidin expression. The quantitative analysis of hepcidin in bodily fluids will provide an insight into the pathogenesis of disorders of iron metabolism such as hereditary hemochromatosis. A mass spectrometry-based approach was taken to detect and quantify urinary hepcidin as urine is readily available and collection of this sample type is non-invasive. Samples were first purified using micro solid phase extraction (μSPE), followed by matrix assisted laser desorption/ionization-orthogonal-time-of-flight mass spectrometry (MALDI-TOF-MS) with inclusion of an internal standard for the quantitative analysis of unlabelled urinary hepcidin. Monoisotopic resolution of hepcidin-25 was observed with the MALDI-TOF-MS and extraction recovery of more than 70% was achieved. Spot-to-spot variations of less than 3.5% RSD and intra- and inter-day precision of less than 9.5% RSD was observed. In our study, average hepcidin-25 levels of 1.7 nmol/mmol creatinine were observed in healthy subjects, compared to an average of 1.1 nmol hepcidin-25/mmol creatinine in hemochromatosis patients. In one of the patients, the hepcidin level was below the detection limit (1.25 nM). From the stability experiments of hepcidin-25, it is recommended that urine samples be stored at -80oC and undergo minimal freeze-thaw cycles. It is also advised that extraction be carried out within 3 hours of thawing. Preliminary analysis using LC-ESI-IT-MS showed multiple charge states of hepcidin-25. However, a variation of less than 5% in hepcidin-25 concentration was observed when the same sample was analyzed with both LC-MS and MALDI-TOF-MS, and thus they can be used to counter-validate each other. In summary, a validated method has been developed for the quantification of unlabelled urinary hepcidin-25 which can be used to investigate the levels of hepcidin-25 in iron-related disorders

Human Milk Lipidomics: Current Techniques and Methodologies

Human milk contains a complex combination of lipids, proteins, carbohydrates, and minerals, which are essential for infant growth and development. While the lipid portion constitutes only 5% of the total human milk composition, it accounts for over 50% of the infant’s daily energy intake. Human milk lipids vary throughout a feed, day, and through different stages of lactation, resulting in difficulties in sampling standardization and, like blood, human milk is bioactive containing endogenous lipases, therefore appropriate storage is critical in order to prevent lipolysis. Suitable sample preparation, often not described in studies, must also be chosen to achieve the aims of the study. Gas chromatography methods have classically been carried out to investigate the fatty acid composition of human milk lipids, but with the advancement of other chromatographic techniques, such as liquid and supercritical fluid chromatography, as well as mass spectrometry, intact lipids can also be characterized. Despite the known importance, concise and comprehensive analysis of the human milk lipidome is limited, with gaps existing in all areas of human milk lipidomics, discussed in this review. With appropriate methodology and instrumentation, further understanding of the human milk lipidome and the influence it has on infant outcomes can be achieved

The Fatty Acid Species and Quantity Consumed by the Breastfed Infant Are Important for Growth and Development

The fatty acids (FAs) of human milk (HM) are the building blocks of the HM lipidome, contributing to infant health and development; however, this has not been comprehensively characterised with respect to infant intake. Eighteen Western Australian mother–infant dyads provided monthly longitudinal HM samples during six months of exclusive breastfeeding. Monthly anthropometric measurements, health data and basic maternal food frequency data were also collected. At three months, infant 24 h milk intake and total lipid intake were measured. The FA profile was analysed using gas chromatography–mass spectrometry. Linear regression and Pearson’s correlation were used to identify associations between HM FA composition, HM FA intake, maternal characteristics and infant growth and developmental outcomes. Mean infant intake of total lipids was 29.7 ± 9.4 g/day. HM FA composition exhibited wide variation between dyads and throughout lactation. Infant intake of a number of FAs, including C15:0, C18:1, C18:2 and C20:3, was positively related to infant growth (all p < 0.001). There were no relationships detected between C22:5 and C20:5 and infant head circumference. Infant total lipid intake and the infant intake of many FAs play essential roles in infant growth and development. This study highlights the important relationships of many HM FAs not previously described, including C15:0 and C18:2 species. Infant outcomes should be considered in the context of intake in future HM studies

Rapid and Self-Administrable Capillary Blood Microsampling Demonstrates Statistical Equivalence with Standard Venous Collections in NMR-Based Lipoprotein Analysis

We investigated plasma and serum blood derivatives from capillary blood microsamples (500 μL, MiniCollect® tubes) and corresponding venous blood (10 mL vacutainers). Samples from twenty healthy participants were analysed by 1H-NMR and 112 lipoprotein subfraction parameters; 3 supramolecular phospholipid composite (SPC) parameters from SPC1, SPC2, and SPC3 subfractions; 2 N-acetyl signals from α-1-acid glycoprotein (Glyc), GlycA and GlycB; and 3 calculated parameters, SPC (total), SPC3/SPC2, Glyc (total)—were assessed. Using linear regression between capillary and venous collection sites, explained variance (Adj. R2 ≥ 0.8, p < 0.001) was witnessed for 86% of plasma parameters (103/120), and 88% of serum parameters (106/120), indicating capillary lipoprotein, SPC, and Glyc concentration follows changes in venous concentra-tions. These results indicate capillary blood microsamples are suitable for sampling in remote areas and for high-frequency longitudinal sampling of the majority of lipoproteins, SPCs, and Glycs

Healthy breastfeeding infants consume different quantities of milk fat globule membrane lipids

10.3390/nu13092951Nutrients139295