"Milk on Ice": A detailed analysis of Ernest Shackleton's century-old whole milk powder in comparison with modern counterparts.

Whole milk powder (WMP) manufactured in New Zealand in 1907 was sent to the Antarctic continent with the Shackleton-led British Antarctic Expedition from 1907 to 1909. This powder was stored at ambient conditions at Shackleton's Hut at Cape Royds, Antarctica, for over 100 yr before a sample was collected on behalf of Fonterra by the Antarctic Heritage Trust. Having spent most of its existence both dried and in frozen storage, any deleterious reactions within the WMP would have been markedly retarded. The composition and some properties of the roller-dried Shackleton's WMP are reported along with those of 2 modern spray-dried New Zealand WMP. The Shackleton powder was less white and more yellow than the modern WMP and was composed of flakes rather than agglomerated particles, consistent with that expected of a roller-dried powder. Headspace analysis showed lipolytic and oxidative volatile compounds were present in the Shackleton WMP, indicting some deterioration of the milk either before powder manufacture or on storage of the finished product. On a moisture-free basis, the Shackleton WMP had higher protein, higher fat (with a markedly higher free fat level), higher ash, and a lower lactose level than the modern WMP. The lysine level was lower in the Shackleton WMP compared with the spray-dried powders, whereas the fatty acid composition was relatively similar. The sodium level was markedly higher in the Shackleton WMP compared with the spray-dried powder, which is probably due to the addition of an alkaline sodium salt to adjust the pH of the milk before roller drying. Lead, iron, and tin levels were markedly higher in the Shackleton WMP compared with the spray-dried powders, possibly due to the equipment used in powder manufacture and the tin-plated cases used for storage. The proteins in the Shackleton WMP were more lactosylated than in the spray-dried powders. The Shackleton WMP had a higher ratio of κ-casein A to B variants and a higher ratio of ß-lactoglobulin B to A variants than the spray-dried powders, whereas the αS1-casein, ß-casein, αS2-casein, and α-lactalbumin protein variants were similar in all powders. The total phospholipid content was markedly lower in the Shackleton WMP than the spray-dried powders, primarily due to a lower phosphatidylethanolamine concentration. The molecular species distributions within the phospholipid classes were generally similar in the 3 powders. Claims are sometimes encountered that the milk of today is different from that consumed by previous generations. However, this comparative study has shown that the Shackleton WMP was generally similar to modern WMP. Although differences in some components and properties were observed, these were attributable to the manufacturing equipment and processes used in the pioneering years of WMP manufacture.

Qualitative and Quantitative Study of Glycosphingolipids in Human Milk and Bovine Milk Using High Performance Liquid Chromatography-Data-Dependent Acquisition-Mass Spectrometry.

Cerebrosides (Crb; including glucosylceramide and galactosylceramide) and lactosylceramide (LacCer) are structurally complex lipids found in many eukaryotic cell membranes, where they play important roles in cell growth, apoptosis, cell recognition and signaling. They are also found in mammalian milk as part of the milk fat globule membrane (MFGM), making milk an important dietary component for the rapidly growing infant. This study reports the development of a robust analytical method for the identification and characterization of 44 Crb and 23 LacCer molecular species in milk, using high performance liquid chromatography-tandem mass spectrometry in data-dependent acquisition mode. For the first time, it also compares the distributions of these species in human and bovine milks, a commercial MFGM-enriched dairy ingredient (MFGM Lipid 100) and commercial standards purified from bovine milk. A method for quantifying Crb and LacCer in milk using mass spectrometry in neutral loss scan mode was developed and validated for human milk, bovine milk and MFGM Lipid 100. Human milk was found to contain approximately 9.9-17.4 µg Crb/mL and 1.3-3.0 µg LacCer/mL, whereas bovine milk (pooled milk from a Friesian herd) contained 9.8-12.0 and 14.3-16.2 µg/mL of these lipids, respectively. The process used to produce MFGM Lipid 100 was shown to have enriched these components to 448 and 1036 µg/g, respectively. No significant changes in the concentrations of both Crb and LacCer were observed during lactation.

Exploring In Vivo Dynamics of Bovine Milk Derived Gangliosides.

Gangliosides are glycosphingolipids present in mammalian cell membranes, playing important structural and functional roles. Human studies on the health benefits of gangliosides are increasing, but knowledge gaps regarding ganglioside analysis exist. The study aimed to investigate blood sample type (serum/plasma), storage conditions, diurnal, day-to-day variation and acute effects of consuming bovine-derived gangliosides on circulating monosialylated gangliosides. Seventy-one women (18-40 yrs, 20-≤30.0 kg/m2) were enrolled and 61 completed the intervention. They visited the clinic three times following overnight fasting. Serum/plasma gangliosides were analyzed over 2 h (visit-1), 8 h (visit-2) and 8 h following either zero or high ganglioside meals (visit-3). Samples stored at -20 °C and -70 °C were analyzed at 3-, 6-, 12- and 18-months. Plasma and serum GM3-gangliosides did not differ, plasma GM3 did not change diurnally, from day-to-day, in response to a high vs. low ganglioside meal or after 7-days low ganglioside vs. habitual diet (P > 0.05). GM3 concentrations were lower in samples stored at -70 °C vs. -20 °C from 6-months onwards and decreased over time with lowest levels at 12- and 18-months stored at -70 °C. In conclusion, either serum/plasma stored at -20- or -70 °C for up to 6 months, are acceptable for GM3-ganglioside analysis. Blood samples can be collected at any time of the day and participants do not have to be in the fasted state.

Human Milk Oligosaccharide, Phospholipid, and Ganglioside Concentrations in Breast Milk from United Arab Emirates Mothers: Results from the MISC Cohort.

Human milk oligosaccharides (HMOs), phospholipids (PLs), and gangliosides (GAs) are components of human breast milk that play important roles in the development of the rapidly growing infant. The differences in these components in human milk from the United Arab Emirates (UAE) were studied in a cross-sectional trial. High-performance liquid chromatography‒mass spectrometry was used to determine HMO, PL, and GA concentrations in transitional (5-15 days) and mature (at 6 months post-partum) breast milk of mothers of the United Arab Emirates (UAE). The results showed that the average HMO (12 species), PL (7 species), and GA (2 species) concentrations quantified in the UAE mothers' transitional milk samples were (in mg/L) 8204 ± 2389, 269 ± 89, and 21.18 ± 11.46, respectively, while in mature milk, the respective concentrations were (in mg/L) 3905 ± 1466, 220 ± 85, and 20.18 ± 9.75. The individual HMO concentrations measured in this study were all significantly higher in transitional milk than in mature milk, except for 3 fucosyllactose, which was higher in mature milk. In this study, secretor and non-secretor phenotype mothers showed no significant difference in the total HMO concentration. For the PL and GA components, changes in the individual PL and GA species distribution was observed between transitional milk and mature milk. However, the changes were within the ranges found in human milk from other regions.

Pasture feeding conventional cows removes differences between organic and conventionally produced milk.

Perceptions of production methods for organic and conventional milk are changing, with consumers prepared to pay premium prices for milk from either certified organic or conventional grass-fed cows. Our study investigated whether chemical composition differed between milk produced by these two farming systems. Sampling was conducted on two farms sets, each comprised of one organic and one conventional farm. All farms applied year-round pasture grazing. Milk samples were collected throughout the milking season and analysed for free oligosaccharides, fatty acids, major casein and whey proteins, and milk fat volatiles. Fatty acids were influenced by breed and fertilizer application. Oligosaccharides differed between farming systems, with causes presently unknown, while farm set was the dominant influence factor on protein composition. Factors identified in this study influencing milk composition are not exclusive to either farming system, and pasture feeding conventional cows will remove differences previously reported for organic and conventionally produced milk.

Dietary supplementation with bovine-derived milk fat globule membrane lipids promotes neuromuscular development in growing rats.

BACKGROUND: The milk fat globule membrane (MFGM) is primarily composed of polar phospho- and sphingolipids, which have established biological effects on neuroplasticity. The present study aimed to investigate the effect of dietary MFGM supplementation on the neuromuscular system during post-natal development. METHODS: Growing rats received dietary supplementation with bovine-derived MFGM mixtures consisting of complex milk lipids (CML), beta serum concentrate (BSC) or a complex milk lipid concentrate (CMLc) (which lacks MFGM proteins) from post-natal day 10 to day 70. RESULTS: Supplementation with MFGM mixtures enriched in polar lipids (BSC and CMLc, but not CML) increased the plasma phosphatidylcholine (PC) concentration, with no effect on plasma phosphatidylinositol (PI), phosphatidylethanolamine (PE), phosphatidylserine (PS) or sphingomyelin (SM). In contrast, muscle PC was reduced in rats receiving supplementation with both BSC and CMLc, whereas muscle PI, PE, PS and SM remained unchanged. Rats receiving BSC and CMLc (but not CML) displayed a slow-to-fast muscle fibre type profile shift (MyHCI → MyHCIIa) that was associated with elevated expression of genes involved in myogenic differentiation (myogenic regulatory factors) and relatively fast fibre type specialisation (Myh2 and Nfatc4). Expression of neuromuscular development genes, including nerve cell markers, components of the synaptogenic agrin-LRP4 pathway and acetylcholine receptor subunits, was also increased in muscle of rats supplemented with BSC and CMLc (but not CML). CONCLUSIONS: These findings demonstrate that dietary supplementation with bovine-derived MFGM mixtures enriched in polar lipids can promote neuromuscular development during post-natal growth in rats, leading to shifts in adult muscle phenotype.

Isotopic labeling of milk disialogangliosides (GD3).

The most abundant ganglioside group in both human milk and bovine milk during the first postnatal week is ganglioside GD3. This group of disialogangliosides forms up to 80% of the total ganglioside content of colostrum. Although dietary gangliosides have shown biological activity such as improvement of cognitive development, gastrointestinal health, and immune function, there is still a gap in our understanding of the molecular mechanisms governing its uptake and the metabolic processes affecting its bioavailability. The use of isotopically labeled ganglioside to track the bioavailability, absorption, distribution, and metabolism of gangliosides may provide key information to bridge this gap. However, isotope labeled GD3 is not commercially available and its preparation has not been described. We report for the first time the preparation of labeled GD3 with stable isotopes. Using alkaline hydrolysis, we were able to selectively remove both acetyl groups from the tetrasaccharide portion of GD3 without promoting significant hydrolysis of the ceramide portion of the molecule to generate N-deacetyl-GD3 (Neu5α2-8Neu5-GD3). The N-deacetyl-GD3 was then chemoselectively re-acetylated in aqueous medium using deuterated acetic anhydride in the presence of Triton X 100 to produce 2H6-GD3 {GD3[(Neu5Ac-11-2H3)-(Neu5Ac-11-2H3)]}. This method provided 2H6-GD3 with approximately 60% yield. This compound was characterized by proton nuclear magnetic resonance (1H NMR) and liquid chromatography mass spectrometry (LC-MS). The oral absorption of the 2H6-GD3 was demonstrated using a Sprague-Dawley weaning rats. Our results indicate that some ingested labeled milk gangliosides are absorbed and transported into the bloodstream without modification.

Ganglioside Composition in Beef, Chicken, Pork, and Fish Determined Using Liquid Chromatography-High-Resolution Mass Spectrometry.

Gangliosides (GA) are found in animal tissues and fluids, such as blood and milk. These sialo-glycosphingolipids have bioactivities in neural development, the gastrointestinal tract, and the immune system. In this study, a high-performance liquid chromatography-mass spectrometry (HPLC-MS) method was validated to characterize and quantitate the GA in beef, chicken, pork, and fish species (turbot, snapper, king salmon, and island mackerel). For the first time, we report the concentration of GM3, the dominant GA in these foods, as ranging from 0.35 to 1.1 mg/100 g and 0.70 to 5.86 mg/100 g of meat and fish, respectively. The minor GAs measured were GD3, GD1a, GD1b, and GT1b. Molecular species distribution revealed that the GA contained long- to very-long-chain acyl fatty acids attached to the ceramide moiety. Fish GA contained only N-acetylneuraminic acid (NeuAc) sialic acid, while beef, chicken, and pork contained GD1a/b species that incorporated both NeuAc and N-glycolylneuraminic acid (NeuGc) and hydroxylated fatty acids.

Effect of Dietary Complex Lipids on the Biosynthesis of Piglet Brain Gangliosides.

Gangliosides, found in mammalian milk, are known for their roles in brain development of the newborn. However, the mechanism involved in the impact of dietary gangliosides on brain metabolism is not fully understood. The impact of diets containing complex lipids rich in milk-derived ganglioside GD3 on the biosynthesis of gangliosides (assessed from the incorporation of deuterium) in the frontal lobe of a piglet model is reported. Higher levels of incorporation of deuterium was observed in the GM1 and GD1a containing stearic acid in samples from piglets fed milk containing 18.2 µg/mL of GD3 compared to that in those fed milk containing 25 µg/mL of GD3. This could suggest that the gangliosides from the diet may be used as a precursor for de novo biosynthesis of brain gangliosides or lead to the reduction of de novo biosynthesis of these gangliosides. This effect was more pronounced in the left compared to that in the right brain hemisphere.

Lactational changes in concentration and distribution of ganglioside molecular species in human breast milk from Chinese mothers.

Gangliosides play a critical role in human brain development and function. Human breast milk (HBM) is an important dietary source of gangliosides for the growing infant. In this study, ganglioside concentrations were measured in the breast milk from a cross-sectional sample of Chinese mothers over an 8-month lactation period. The average total ganglioside concentration increased from 13.1 mg/l during the first month to 20.9 mg/l by 8 months of lactation. The average concentration during the typically solely breast-feeding period of 1‒6 months was 18.9 mg/l. This is the first study to report the relative distribution of the individual ganglioside molecular species through lactation for any population group. The ganglioside molecular species are made up of different fatty acid moieties that influence the physical properties of these gangliosides, and hence affect their function. The GM(3) molecular species containing long-chain acyl fatty acids had the most prominent changes, increasing in both concentration and relative distribution. The equivalent long-chain acyl fatty acid GD(3) molecular species typically decreased in concentration and relative distribution. The lactational trends for both concentration and relative distribution for the very long-chain acyl fatty acid molecular species were more varied. The major GM(3) and GD(3) molecular species during lactation were d40:1 and d42:1, respectively. An understanding of ganglioside molecular species distribution in HBM is essential for accurate application of mass spectrometry methods for ganglioside quantification.

Long-Term Supplementation with Beta Serum Concentrate (BSC), a Complex of Milk Lipids, during Post-Natal Brain Development Improves Memory in Rats.

We have previously reported that the supplementation of ganglioside-enriched complex-milk-lipids improves cognitive function and that a phospholipid-enriched complex-milk-lipid prevents age-related cognitive decline in rats. This current study evaluated the effects of post-natal supplementation of ganglioside- and phospholipid-enriched complex-milk-lipids beta serum concentrate (BSC) on cognitive function in young rats. The diet of male rats was supplemented with either gels formulated BSC (n = 16) or blank gels (n = 16) from post-natal day 10 to day 70. Memory and anxiety-like behaviors were evaluated using the Morris water maze, dark-light boxes, and elevated plus maze tests. Neuroplasticity and white matter were measured using immunohistochemical staining. The overall performance in seven-day acquisition trials was similar between the groups. Compared with the control group, BSC supplementation reduced the latency to the platform during day one of the acquisition tests. Supplementation improved memory by showing reduced latency and improved path efficiency to the platform quadrant, and smaller initial heading error from the platform zone. Supplemented rats showed an increase in striatal dopamine terminals and hippocampal glutamate receptors. Thus BSC supplementation during post-natal brain development improved learning and memory, independent from anxiety. The moderately enhanced neuroplasticity in dopamine and glutamate may be biological changes underlying the improved cognitive function.

Maternal serum and breast milk vitamin D levels: findings from the Universiti Sains Malaysia Pregnancy Cohort Study.

BACKGROUND: Vitamin D deficiency has become a global health issue in pregnant women. This study aimed to assess the adequacy of maternal vitamin D status by measuring maternal serum and breast milk 25-hydroxyvitamin D [25(OH)D] levels and to determine the association between maternal serum and milk 25(OH)D levels. METHODS: Data was obtained from the Universiti Sains Malaysia Pregnancy Cohort Study. This study was conducted from April 2010 to December 2012 in the state of Kelantan, Malaysia. Blood samples from pregnant women aged 19 to 40 years were drawn in the second and third trimesters of pregnancy, while breast milk samples at delivery, 2, 6 and 12 months postpartum were collected to analyze for 25(OH)D levels. A total of 102 pregnant women were included in the analysis. RESULTS: Vitamin D deficiency [25(OH)D <50 nmol/L] was detected in 60% and 37% of women in the second and third trimesters of pregnancy, respectively. There were 6% and 23% of women who reached normal level of vitamin D status in the second trimester and the third trimester, respectively. Multivitamin intakes during pregnancy were significantly associated with higher serum 25(OH)D levels in the second trimester (ß = 9.16, p = 0.005) and the third trimester (ß = 13.65, p = 0.003). 25(OH)D levels in breast milk during the first year of lactation ranged from 1.01 to 1.26 nmol/L. Higher maternal serum 25(OH)D level in the second trimester of pregnancy was associated with an elevated level of 25(OH)D in breast milk at delivery (ß = 0.002, p = 0.026). CONCLUSIONS: This study shows that high proportions of Malay pregnant women are at risk of vitamin D deficiency. Maternal vitamin D status in the second trimester of pregnancy was found to influence vitamin D level in breast milk at delivery.

Analysis of phospholipids in infant formulas using high performance liquid chromatography-tandem mass spectrometry.

Phospholipids have been used widely in the food industry as emulsifiers, but it is their biological and nutritional function that has been the focus over recent years. The recognition of the importance of phospholipids and sphingomyelin for infant development has led to an increase in the number of infant formulas claiming to contain these complex lipid components. Therefore, the ability to measure these lipids in infant formulas and dairy-derived complex lipid ingredients for fortification purposes is important. A high-performance liquid chromatography-tandem mass spectrometry method that quantifies phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, and sphingomyelin found in infant formulas and dairy-derived complex lipid ingredients is described. The method uses external standards of similar fatty acid profile for calibration. The recovery of phospholipids ranged from 92% to 102% with a method repeatability coefficient of variation of 6-10%. In addition to the specificity and selectivity of the method, details of the molecular species in the individual phospholipid classes are available using this method.

Quantification of bovine milk oligosaccharides using liquid chromatography-selected reaction monitoring-mass spectrometry.

Oligosaccharides are important components of milk with bioefficacy as prebiotics, anti-infectives, and immune system modulators and as a possible source of sialic acid for neural function. Bovine milk oligosaccharides are lower in concentration and lack the diversity of human milk oligosaccharides but could be a commercial source of milk oligosaccharides for pediatric foods. For this development, an ability to quantify the oligosaccharides is required. This study validated a hydrophilic interaction chromatography high-performance liquid chromatography-high-resolution selected reaction monitoring-mass spectrometry (HILIC HPLC-HRSRM-MS) method for measuring six different oligosaccharides in bovine milk, bovine colostrum, and infant formulas. The extraction resulted in a high recovery (90-103%) with a repeatability coefficient of variation ranging from 2 to 9% for the two dominant oligosaccharides, 3'-sialyllactose and 6'-sialyllactose, and ranging from 1 to 17% for the much lower concentration oligosaccharides, 6'-sialyllactosamine, disialyllactose, and N-acetylgalactosaminyllactose. The sixth oligosaccharide, 3'-sialyllactosamine, was not detected in any of the samples.

Maternal supplementation with a complex milk lipid mixture during pregnancy and lactation alters neonatal brain lipid composition but lacks effect on cognitive function in rats.

Complex milk lipids (CMLs) provide a critical nutritional source for generating both energy and essential nutrients for the growth of the newborn. The present study investigated nutritional supplementation with a CML containing gangliosides and phospholipids in pregnant and lactating rats on learning behavior and postnatal growth in male offspring. Wistar female rats were supplemented during pregnancy and lactation with either control or CML to provide gangliosides at a dose of 0.01% (low) and 0.05% (high) based on total food intake. The CML-supplemented dams showed no differences in comparison to controls regarding growth, food intake, and litter characteristics. There were significant differences in brain composition in male offspring at postnatal day 2 (P2) with higher concentrations of gangliosides (high dose, P < .05) and lower concentrations of phospholipids (low and high dose, P < .05) in the CML-supplemented groups. The distribution of individual ganglioside species was not significantly different between treatment groups. Brain weight at P2 was also significantly higher in the CML groups. Differences in the brain composition and weight were not significant by weaning (P21). As adults (P80), adiposity was reduced in the low CML-supplemented group compared to controls. No significant differences were detected between any of the treatment groups in any of the behavioral tasks (water maze, object recognition, and operant learning). These data suggest that maternal supplementation with a CML during pregnancy and lactation is safe and has a significant early impact on brain weight and ganglioside and phospholipid content in offspring but did not alter long-term behavioral function using standard behavioral techniques.

Analysis of phospholipids in rat brain using liquid chromatography-mass spectrometry.

The brain is a lipid-rich organ containing complex polar lipids including phospholipids (PLs) and sphingolipids. These lipids are involved in the structure and function of cell membranes in the brain. We developed a fast and efficient liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify five different classes of PLs [Choline glycerophospholipid (consists of phosphatidyl choline and plasmenyl choline in these samples), ethanolamine glycerophospholipid (consist of phosphatidyl ethanolamine and plasmenyl ethanolamine in these samples), phosphatidyl serine, phosphatidyl inositol, and sphingomyelin] in the brain tissues of 80-day-old Wistar rats. The PLs were extracted from rat brain using chloroform/methanol/water. After separation using a hydrophilic high performance liquid chromatography column, PL-class-specific fragmentation (head group identification) with a tandem mass spectrometer in positive ion mode was utilized to measure changes in the relative concentration of the five PL classes. The advantage of this approach was its improved specificity over previously reported LC-MS methods. The method had good repeatability (coefficient of variation 3-9%, excluding phosphatidyl inositol) and recovery (92-103%) and compared well with more laborious traditional methods.

Liquid chromatography-high-resolution mass spectrometry for quantitative analysis of gangliosides.

Gangliosides are a large family of glycosphingolipids that are abundant in the brain, and have been shown to affect neuronal plasticity during development, adulthood, and aging. We developed a fast, efficient, and sensitive liquid chromatography-mass spectrometry method to quantify eight different classes of gangliosides (GM(1), GM(2), GM(3), GD(3), GD(1a), GD(1b), GT(1b), GQ(1b)) in the brains of 2-day-old and 80-day-old Wistar rats. The gangliosides were extracted from rat brain using a modified Svennerholm and Fredman method. After ganglioside class separation using a hydrophilic high performance liquid chromatography (HPLC) column, the resolving power of the LTQ-Orbitrap mass spectrometer was used to extract and sum the major species of each ganglioside class, generating fully resolved extracted ion current peaks for both standards and samples. The flexibility and the specificity of this method are such that it can be applied to the analysis of other ganglioside species/classes not discussed in this paper, provided appropriate standards are available. The method had good repeatability (coefficient of variation 4.8-12.3%) and mean recoveries in the range 92-107%.

Supplementation with a mixture of complex lipids derived from milk to growing rats results in improvements in parameters related to growth and cognition.

Alterations in nutritional factors during early development can exert long-term effects on growth, neural function, and associated behaviors. The lipid component of milk provides a critical nutritional source for generating both energy and essential nutrients for the growth of the newborn. The present study, therefore, investigated the hypothesis that nutritional supplementation with a complex milk lipid (CML) preparation, derived from the milk fat globule membrane rich in phospholipids and gangliosides from young rats, has beneficial effects on learning behavior and postnatal growth and development. Male Wistar rat offspring from normal pregnancies were treated from neonatal day 10 until postnatal day 80 with either vehicle or CML at a dose of 0.2% (low) and 1.0% (high) based on total food intake (n = 16 per group). Neonatal dosing was via daily oral gavage, while postweaning dosing was via gel supplementation to a standard chow diet. Animals underwent behavioral tasks related to spatial memory, learning, and cognitive function. Complex milk lipid supplementation significantly increased linear growth rate (P < .05), and the improved growth trajectory was not related to changes in body composition as quantified by dual-energy x-ray absorptiometry scanning or altered plasma lipid profiles. Moreover, this effect was not dose dependent and not attributable to the contribution to total energy intake of the CML composition. Supplementation of the CML to growing rats resulted in statistically significant improvements in parameters related to novelty recognition (P < .02) and spatial memory (P < .05) using standard behavioral techniques, but operant testing showed no significant differences between treatment groups. Supplementation with a CML containing gangliosides had positive growth and learning behavioral effects in young normal growing rats.

Quantification of milk fat globule membrane proteins using selected reaction monitoring mass spectrometry.

Although some of the physiological roles of milk fat globule membrane (MFGM) proteins are still unclear, there is increasing evidence that the consumption of bovine MFGM proteins has significant nutritional health benefits for humans; therefore, it may be important to be able to estimate the MFGM proteins in complex ingredients. In this study, the absolute quantification (AQUA) technique, which is typically used for the quantification of proteins in proteomic studies, was applied for the quantification of bovine MFGM proteins in butter milk protein concentrate. Six MFGM proteins (fatty acid binding protein, butyrophilin, PAS 6/7, adipophilin, xanthine oxidase, and mucin 1) were simultaneously quantified using high-resolution selected reaction monitoring mass spectrometry. Samples were rehydrated in 6.7 M urea buffer prior to dilution to 2.2 M before tryspin digestion. Direct rehydration in 2.2 M urea buffer or 2.2 M urea/20% acetonitilrile buffer reduced peptide yield digestion. Isotopically labeled peptides were used as internal standards. The coefficient of variation ranged from 5 to 15%, with a recovery of 84-105%. The limit of detection was in the range of 20-40 pg.

Determination of vitamin K1 isomers in foods by liquid chromatography with C30 bonded-phase column.

Vitamin K1 was determined in a variety of foods by using reversed-phase liquid chromatography with a C30 column followed by post-column reduction to the fluorescent hydroquinone derivatives. Lipids were removed by lipase digestion, followed by single extraction into hydrocarbon, and the protocol was extended to selected natural and processed foods. Biologically active trans- and inactive ci-vitamin K1 isomers were measured individually to evaluate the true nutritional status of the products. Method performance parameters confirmed the validity of the technique. The use of the triacontyl-bonded C30 phase for selective phylloquinone isomer measurement extends previously validated AOAC Method 999.15 for vitamin K1 in milk and infant formula to a wider range of foods important in the human diet. The cis-vitamin K1 isomer contributes up to about 15% of total phylloquinone in certain foods.