A Collection of Articles from LIFE Research

Editor(s): Various.


A new method for determination of human milk oligosaccharides (HMO)

Author(s): Austin S, Bénet T.

Quantitative determination of non-lactose milk oligosaccharides.

Non-lactose oligosaccharides (NLO) in milk vary across species, while they are not very abundant in cow milk, the concentration of human milk oligosaccharides (HMO) can reach as high as 20g/l. They are not completely absorbed in the infant’s digestive tract, and while their role is still being studied some of their benefits include preventing pathogens from binding to the host cell surfaces, and acting as substrates for developing the infant’s gut microbiome.  Many different approaches have been used for the quantitative determination of HMO, however results are not well comparable between studies, methods are generally poorly validated (if at all), and accessing good quality HMO standards is very difficult. This work describes a methodology based on labelling the HMO with a fluorescent tag and analyzing  the tagged HMO by  liquid chromatography with fluorescence detection. The method offers several advantages including: a) it does not require extensive purification of oligosaccharides prior to analysis; b) by adding a fluorescent tag to HMOs, it enables the quantitative determination of the oligosaccharides in the absence of the HMO standard; c) it allows the determination of a large number of HMOs; d) it has been extensively validated.

A new method for determination of gangliosides in human milk and their concentration across lactation

Author(s): Giuffrida F, Elmelegy IM, Thakkar SK, Marmet C, Destaillats F.

Longitudinal evolution of the concentration of gangliosides GM3 and GD3 in human milk

Gangliosides are glycosphingolipids formed by a ceramide and an oligosaccharide chain; they are distributed in most human tissues with the highest amount found in neural tissue, lung, spleen and gut. During early life dietary gangliosides may be have an important role on brain development, modification of the intestinal microflora and promotion of intestinal immunity. While their presence in human milk is known, quantitative data on their concentration is still scarce.

The diverse structures in the lipid moiety and in the oligosaccharide originate different gangliosides, and this work describes a validated procedure to quantify the two major classes: GD3 and GM3.  Previously existing methods had either low sensitivity or were very time consuming limiting the amount of samples that could be analyzed. The method described in this paper uses liquid chromatography coupled with electrospray ionization high-resolution mass spectrometer and it has several advantages including: more selectiveness and robustness, allows the identification of molecular species of GD3 and GM3 and it is feasible to use in large sample sets.

The authors were able to provide the first report of longitudinal ganglioside concentrations in human milk. They analyzed a large cohort with samples at 0-11, 30, 60 and 120 days after birth. The content of GD3 and GM3 in human milk ranged between 0.9 to 3.8 µg/ml and 4.3 to 9.8 µg/ml, respectively. At the beginning of the lactation period (0-11 days) the amounts of GD3 and GM3 were comparable and the ratio GM3:GD3 was 1:1. Thereafter GM3 concentration increased and GD3 decreased, and the ratio GM3:GD3 was 10:1 at 60 and 120 days. Assuming a mean volume of breast milk consumption of 600, 700 and 800 ml, the estimated intake of ganglioside in infants is 5.5, 7.0 and 8.6 mg/d at 30, 60 and 120 d postpartum.  Further investigations are needed to determine the biological function of gangliosides in milk.

Lipids. 2014 Oct;49(10):997-1004. doi: 10.1007/s11745-014-3943-2.

Fatty acids in preterm milk may differ from term milk

Author(s): Thakkar SK, De Castro CA, Beauport L, Tolsa JF, Fischer Fumeaux CJ, Affolter M, Giuffrida F.

Temporal Progression of Fatty Acids in Preterm and Term Human Milk of Mothers from Switzerland

In preterm infants, human milk reduces morbidity, mortality, and enhances neurodevelopment. Because of their benefits, it should be the primary choice for feeding, however, fortification of maternal milk might be necessary in order to meet the high nutritional demands form mimicking intrauterine growth. Lipids provide about 50% of total energy in human milk and are present mostly as triacylglycerols, formed by one glycerol and three fatty acids, which range from medium to very long chain and may be saturated, mono-unsaturated and poly-unsaturated. Human milk provides essential fatty acids and bioactive fatty acids among others.  

This study analyzed fatty acids in colostrum, transitional and mature milk of mothers delivering preterm infants (28-32 weeks gestational age) and compared to milk from mothers delivering term infants (>37 weeks gestational age), the study was done in Switzerland and included 61 mothers.

Total lipids increased as lactation advanced, but mature milk of the preterm had lower concentration of total lipids. Colostrum for preterm infants had greater concentration of medium chain fatty acids (caprylic 8:0, capric 10:0 and lauric 12:0), as well as greater α linolenic acid (ALA, 18:3 n-3), but was lower in dihomo ƴ linolenic (20:3 n-6) , erucic (20:3 n-6) and nervonic acid (24:1 n-9). Compared to term infants mature milk for preterm infants had more saturated fatty acids, and specifically lauric (12:0) and myristic (14:0) acids, as well as more linoleic acid (LA, 18:2 n-6), and less monounsaturated fatty acids (MUFA). There were no differences between preterm and term groups in total polyunsaturated fatty acids and neither in DHA (22:6 n-3), ARA (20:4 n-6) and EPA (20:5 n-3) content neither in colostrum nor in mature milk. This work provides evidence of different composition in the fatty acids of preterm milk compared to term milk, although their implication on developmental outcomes and fortification strategies remain to be investigated.

Breast milk microbiota of Chinese mothers

Author(s): Sakwinska O, Moine D, Delley M, Combremont S, Rezzonico E, Descombes P, Vinyes-Pares G, Zhang Y, Wang P, Thakkar SK

Microbiota in Breast Milk of Chinese Lactating Mothers

Numerous studies have reported the presence of bacteria in breast milk. Although the initial focus was on detection of potential pathogens, there is increasing evidence that the presence of bacteria in different niches of the human body is the physiological norm. Therefore, there is interest in characterizing milk microbiota and studying their potential benefits for the breastfed infant. Previous data obtained from American and European women suggest that geographical and ethnical differences may play a role on milk microbiota composition, in addition, the methodologies used for analysis and milk collection can account for some variability. Most studies have collected milk in aseptic conditions, and only a few have analyzed samples collected by standard methods (without previous cleansing).

This study explored the microbiota from Chinese mothers at three time points within the first 2 months of lactation and compared samples collected with aseptic vs standard procedures. The total amount of bacteria was higher with standard than in aseptic collection. In accordance to what has been reported in other geographical regions, staphylococci and streptococci dominated breast milk microbiota from Chinese women and constituted 42 and 40% of total bacteria. Acinetobacter sp was very abundant in standard collection samples representing an average of 32% of the total, but not by aseptic procedures where it was only 1.8%. Lactobacilli and bifidobacteria were present only in few samples with low abundance (less than 1%) in both, aseptic and standard collected samples.  

There was no impact on delivery mode or stage of lactation in the samples studied. This study provides further evidence on the presence of bacteria in human milk and data on the milk microbiota of Chinese women. In addition, the authors suggest the name ‘Breastfeeding-associated microbiota’ to that found in standard collected samples which is more representative of the microbiota that the infant ingest.

Carotenoids and tocopherols concentrations in breast milk

Author(s): Xue Y, Campos-Giménez E, Redeuil KM, Lévèques A, Actis-Goretta L, Vinyes-Pares G, Zhang Y, Wang P, Thakkar SK.

Concentrations of Carotenoids and Tocopherols in Breast Milk from Urban Chinese Mothers and Their Associations with Maternal Characteristics: A Cross-Sectional Study

Vitamin A and D are required in many metabolic functions for the growing infant, and are present in breast milk in the form of preformed retinol and carotenoids (Vit A) and tocopherols (Vit E). 

This study analyzed carotenoids (β carotene, β crypptoxanthin, lutein, lycopene and zeaxanthin) and tocopherols (α and ƴ tocopherol) in breast milk taken between 4 and 240 days postpartum in a cross-sectional study in China.  Most of the carotenoids and tocopherols were higher in early lactation compared to later time points. While there were no significant associations with dietary intake of vitamin A and E, other maternal factors like mode of delivery, BMI and maternal education seem to be associated to levels of zeaxanthin and β carotene. Given the importance of these nutrients on early infant development, more research should be conducted to understand maternal factors that affect them.

Amino acid composition of breast milk

Author(s): Garcia-Rodenas CL, Affolter M, Vinyes-Pares G, De Castro CA, Karagounis LG, Zhang Y, Wang P, Thakkar SK

Protein in breast is a key nutrient for supporting growth and development; it provides indispensable amino acids for protein building and can stimulate secretion of growth-promoting factors.  Most amino acids in breastmilk are constituents of proteins, but there is a small amount found as free amino acid (FAA).  This study analyzed total amino acids (TAA) and free amino acids (FAA) in breastmilk of Chinese mothers covering eight months of lactation. TTA decreased as lactation progresses, and the concentration of indispensable and dispensable amino acids was highest in early milk samples. Leucine   and methionine were the most and the least abundant indispensable amino acids, respectively. FFA contributed to less than 2% of TTA, and were lower in early stages compared to later. Glutamic acid was the most abundant FAA contributing to 70% of the mass. The amino acid composition of breast milk in this study is comparable with other reports suggesting that it is an evolutionary conserved trait.

Nutrients. 2016 Sep 28;8(10). pii: E606

Temporal changes of protein composition and impact of caesarean section

Author(s): Affolter M, Garcia-Rodenas CL, Vinyes-Pares G, Jenni R, Roggero I, Avanti-Nigro O, de Castro CA, Zhao A, Zhang Y, Wang P, Thakkar SK, Favre L

In addition to providing amino acids for growth, breast milk protein also has immunologically active molecules that confer passive immunity, stimulate antimicrobial defenses and promote immune maturation.  The most abundant proteins in breast milk are lactoferrin, α-lactalbulmin, serum albumin and the β and κ casein fractions, which represent about 85% of total protein, while immune factors like IgA, IgM, IgG, TGFβ1 and TGFβ2 represent about 10%.  Infants born by C-section had greater risk of developing immune-related diseases and this has been attributed to altered microbiota colonization, but altered immune factors in breast milk may also contribute.  Therefore, this work assessed the main changes in breast milk proteins and assessed the impact of delivery by C-section in the MING study, a cross sectional study that took place in China.

Both, α-lactalbulmin and lactoferrin concentrations showed a temporal pattern and were higher at early stages of lactation, while serum albumin concentrations were constant across lactation. Casein was higher in the samples taken between 12 day and 2 months and decreased thereafter. Immune factors changed according to the stage of lactation: concentrations of IgA and IgM were higher in early milk and then rapidly decreased reaching a plateau by 1 month; and TGFβ1 and TGFβ2 were higher at the beginning and then decreased. C-section did not seem to affect immune factors concentration.

Nutrients. 2016 Aug 17;8(8). pii: E504. doi: 10.3390/nu8080504.

Human milk oligosaccharides and infant growth

Author(s): Sprenger N, Lee LY, De Castro CA, Steenhout P, Thakkar SK

Human milk oligosaccharides are one of the most abundant components of breast milk. They are formed by lactose and N-acetyl glucosamine, galactose, sialic acid or fucose.  The enzymes that transfer fucose are determined by maternal genotype, and the genes FUT2 and FUT3 are responsible for the Secretor negative (FUT2-/-) and Lewis negative (FUT3-/-) glycosylation phenotypes. In women with functional FUT2 the major milk oligosaccharides are 2FL, LNFP1 and diFL. Some research has linked maternal secretor status to early establishment of bifidobacteria in the infant’s microbiota, which could have implications for infant growth and immune development. The objective of this work was to characterize the longitudinal changes in FUT2-dependent HMOs across lactation and explore association to infants’ early growth. The study took place in Singapore and included 50 mother-infant pairs during the first 4 months of lactation, which were divided in two groups: high or low 2FL in breastmilk as a proxy for secretor status.  LNT was the most prominent HMO in the low 2FL group, the content of 2FL, LNT, LnNT and 6’SL decreased across lactation while 3’SL remained constant.  In this cohort with healthy term infants, there were no differences in growth z-scores between groups consuming milk with low or high FUT2.

PLoS One. 2017 Feb 9;12(2):e0171814. doi: 10.1371/journal.pone.0171814. eCollection 2017.

Method for quantification of glycerophospholipids and sphingomyelin in human milk and infant formula

Author(s): Tavazzi I, Fontannaz P, Lee LY, Giuffrida F

Lipids provide 50% of the calories in human milk and are present in the form of fat globules, which are made of triacylglycerols surrounded by a membrane composed of phospholipids, cholesterol, enzymes, proteins, glycosphingolipids and glycoproteins. Phospholipids and sphingomyelin are difficult to analyze quantitatively in breast milk due to their low amounts and their chemical properties (they contain both: a water-soluble and a water-insoluble group). Previous methods described in the literature were either only adequate for qualitative and semi-quantitative purposes or were costly and required highly trained operators, thus not suitable for analyzing a large set of samples. This works describes a method validation to quantify the most abundant phospholipid classes in human milk using high performance liquid chromatography coupled with mass spectrometer detector (HPLC-MS/MS). The method was used successfully also to analyze infant formula demonstrating feasibility for application to other matrixes.

J Chromatogr B Analyt Technol Biomed Life Sci. 2018 Jan 1;1072:235-243. doi: 10.1016/j.jchromb.2017.10.067. Epub 2017 Nov 7

Comparison of macronutrient content in human milk measured by mid-infrared human milk analyzer and reference methods

Author(s): Giuffrida F, Austin S, Cuany D, Sanchez-Bridge B, Longet K, Bertschy E, Sauser J, Thakkar SK, Lee LY, Affolter M.

Lipids, carbohydrates and proteins are the major components of human milk.  Conventional analysis of these components require individual methodologies and a large milk volume. While these methods are sensitive and accurate, they are not adequate for clinical application. Preterm babies are often fed with mother’s own milk fortified with single or multiple nutrients, therefore rapid analysis of milk macronutrient content is key for personalized nutrition. Human milk analyzers (HMA) were developed for use in hospital settings; they require small milk volume and are based on mid-infrared transmission spectroscopy. This study compared the results obtained with MIRIS-HMA with the values obtained by the reference methods (Rose-Gottlieb, HPAEC-PAD and Kjeldahl for fat, lactose, and protein, respectively).  The amount of lactose determined with the HMA was not different from the reference method; however, there was a difference of 12% in fat, which was within the variability reported by the supplier of the MIRIS-HMA. For protein, the difference was more than 15% and therefore the authors screened another rapid method for protein content determination in small volumes- They found that the BCA protein assay was not significantly different from the reference method and may be more appropriate for milk protein analysis.

J Perinatol. 2018 Dec 14. doi: 10.1038/s41372-018-0291-8.

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