MALDI Q-TOF CID MS for Diagnostic Ion Screening of Human Milk Oligosaccharide Samples

Human milk oligosaccharides (HMO) represent the bioactive components of human milk, influencing the infant’s gastrointestinal microflora and immune system. Structurally, they represent a highly complex class of analyte, where the main core oligosaccharide structures are built from galactose and N-acetylglucosamine, linked by 1–3 or 1–4 glycosidic linkages and potentially modified with fucose and sialic acid residues. The core structures can be linear or branched. Additional structural complexity in samples can be induced by endogenous exoglycosidase activity or chemical procedures during the sample preparation. Here, we show that using matrix-assisted laser desorption/ionization (MALDI) quadrupole-time-of-flight (Q-TOF) collision-induced dissociation (CID) as a fast screening method, diagnostic structural information about single oligosaccharide components present in a complex mixture can be obtained. According to sequencing data on 14 out of 22 parent ions detected in a single high molecular weight oligosaccharide chromatographic fraction, 20 different oligosaccharide structure types, corresponding to over 30 isomeric oligosaccharide structures and over 100 possible HMO isomers when biosynthetic linkage variations were taken into account, were postulated. For MS/MS data analysis, we used the de novo sequencing approach using diagnostic ion analysis on reduced oligosaccharides by following known biosynthetic rules. Using this approach, de novo characterization has been achieved also for the structures, which could not have been predicted.     

Role of polyamines in diet. Importance of polyamines in infant nutrition

The first studies indicating polyamines as important growth factors in breast milk began during the nineties of last century. Nevertheless, it is still not well known the role they play in infant nutrition or what the recommended intake would be for this population group. In recent years, there has been increased attention of the international scientific community towards polyamines, not only due to the important role they play in the cellular metabolism, but also to their possible implication in some diseases and during the development of the human organism. Bearing in mind that the content in polyamines of the infant formula is around tenfold less than in breast milk, it would be recommended to gain insight into this theme in order to guarantee correct nutrition during lactation.     

葡萄糖、丁酸钠和乳酸对重组CHO细胞表达EPO糖基化的影响

目的 研究葡萄糟、丁酸钠和乳酸对ＣＨＯ细胞表达的重组人红细胞生成素（ＥＰＯ）糖基化的影响。 方法 将重组ＣＨＯ细胞在添加一定浓度的葡萄糖、丁酸钠和乳酸的培养基中培养,从收获上清中分离纯化ＥＰＯ,定 量测定其唾液酸含量和分析其糖基化位点占有率。结果 在实验范围内,葡萄槽对糟基化影一较小;丁酸钠明显 降低了唾液酸含量,但使Ｎ－连接寡糖的位点占有卒略有增加;Ｌ－乳酸的作用相反,高浓度的乳酸增加了唾液酸 含量,Ｎ－连接寡糖的位点占有率略有降低。结论 丁酸钠和乳酸对重组ＣＨＯ细胞表达的人ＥＰＯ的糖基化有明显 影响,葡萄糖的作用较小。     

Lipids and human milk

To support the growth and development of the breast‐fed infant, human milk provides the dietary essential fatty acids, linoleic acid (LA; 18:2n‐6), α‐linolenic acid (ALA, 18:3n‐3), as well as longer‐chain polyunsaturated fatty acids including arachidonic acid (20:4n‐6) and docosahexanoic (DHA 22:6n‐3). The linoleic acid, alpha‐linolenic acid, DHA and arachidonic acid concentration of pasteurized and unpasteurized human milk remains stable during the first month of storage at –20°C and –80°C. However after the first month, a slow decrease in concentration progresses until the end of 6 months of storage at both temperatures. The levels of n‐6 and n‐3 fatty acids, particularly linoleic acid, alpha‐linolenic acid and DHA, in human milk vary widely within and among different populations, and are readily changed by maternal dietary intake of the respective fatty acid. The present paper reviews recent understanding from key researchers of maternal diet and human milk fat composition and form our work the effect of milk fat composition on storage conditions. It is important to understand that maternal diet can affect human milk fat composition and subsequently infant development and growth.     

Polyunsaturated fatty acids and T-cell function: Implications for the neonate

Infant survival depends on the ability to respond effectively and appropriately to environmental challenges. Infants are born with a degree of immunological immaturity that renders them susceptible to infection and abnormal dietary responses (allergies). T-lymphocyte function is poorly developed at birth. The reduced ability of infants to respond to mitogens may be the result, of the low number of CD45RO+ (memory/antigen-primed). T cells in the infant or the limited ability to produce cytokines [particularly interferon-γ, interleukin (IL)-4, and IL-10]. There have been many important changes in optimizing breast milk substitutes for infants; however, few have been directed at replacing factors in breast milk that convey immune benefits. Recent research has been directed at the neurological, retinal, and membrane benefits of adding 20∶4n−6 (arachidonic acid; AA) and 22∶6n−3 (docosahexaenoic acid; DHA) to infant formula. In addults and animals, feeding DHA affects T-cell function. However, the effect of these lipids on the development and function of the infant's immune system is not known. We recently reported the effect of adding DHA+AA to a standard infant formula on several functional indices of immune development. Compared with standard formula, feeding a formula containing DHA+AA increased the proporition of antigen mature (CD45RO+) CD4⁺ cells, improved IL-10 production, and reduced IL-2 production to levels not different from those of human milk-fed infants. This review will briefly describe T-cell development and the potential immune effect of feeding long-chain polyunsaturated fatty acids to the neonate.     

Human Milk Oligosaccharides and Bacterial Profile Modulate Infant Body Composition during Exclusive Breastfeeding

Human milk is a complex and variable ecosystem fundamental to the development of newborns. This study aimed to investigate relationships between human milk oligosaccharides (HMO) and human milk bacterial profiles and infant body composition. Human milk samples (n = 60) were collected at two months postpartum. Infant and maternal body composition was measured with bioimpedance spectroscopy. Human milk bacterial profiles were assessed using full-length 16S rRNA gene sequencing and 19 HMOs were quantitated using high-performance liquid chromatography. Relative abundance of human milk bacterial taxa were significantly associated with concentrations of several fucosylated and sialylated HMOs. Individual human milk bacteria and HMO intakes and concentrations were also significantly associated with infant anthropometry, fat-free mass, and adiposity. Furthermore, when data were stratified based on maternal secretor status, some of these relationships differed significantly among infants born to secretor vs non-secretor mothers. In conclusion, in this pilot study the human milk bacterial profile and HMO intakes and concentrations were significantly associated with infant body composition, with associations modified by secretor status. Future research designed to increase the understanding of the mechanisms by which HMO and human milk bacteria modulate infant body composition should include intakes in addition to concentrations.     

The chemistry and biology of trypanosomal trans-sialidases: virulence factors in Chagas disease and sleeping sickness

trans-Sialidases constitute a special group of the sialidase family. They occur in some trypanosome species and, in a unique reversible reaction, transfer sialic acids from one glycosidic linkage with galactose (donor) to another galactose (acceptor), to form (α2-3)-sialyl linkages. Trypanosomes cause such devastating human diseases as Chagas disease in South America (Trypanosoma cruzi) or sleeping sickness in Africa (Trypanosoma brucei). The trans-sialidases strongly contribute to the pathogenicity of the trypanosomes by scavenging sialic acids from the host or blood meal to coat the parasite surface; this aids their survival strategy in the insect's intestine, and in the blood circulation or cells of the host, and serves to compromise the immune system of the human or animal host. American and African trypanosomes express trans-sialidases at different stages of their vector/host development. They are transmitted to humans by insect vectors (tsetse fly or other insect "bug" species). trans-Sialidase activity with varying linkage specificity has also been found in a few bacteria species and in human serum. trans-Sialidases are of increasing practical importance for the chemo-enzymatic synthesis of sialylated glycans. The search for appropriate inhibitors of trans-sialidases and vaccination strategies is intensifying, as less toxic medicaments for the treatment of these widespread and often chronic tropical diseases are required.     

Supplemental Conjugated Linoleic Acid Consumption Does Not Influence Milk Macronutrient Contents in all Healthy Lactating Women

The term “conjugated linoleic acid” (CLA) refers to a group of positional and geometric isomers that are derived from linoleic acid and are found primarily in meat and milk products from ruminant animals. Due to the array of putative benefits associated with various forms of CLA, there has been recent interest in supplementing human diets with these fatty acids especially when weight loss is desired. However, in many animal models, CLA has been shown to decrease milk fat production. There is some concern, therefore, that maternal CLA supplementation during lactation might inadvertently decrease nutrient supply to the nursing infant. However, there is only limited research on the effect of CLA consumption on milk fat content in women. Based on previously published work from our laboratory, we hypothesized that CLA supplementation would reduce the milk fat percentage in lactating women in a dose-dependent manner. Breastfeeding women (n = 12) were assigned randomly to treatments of 4 g/day safflower oil (SFO), 2 g/day CLA plus 2 g/day SFO, or 4 g/day CLA in a double blind, 3 × 3 Latin square design. Conjugated linoleic acid supplements contained approximately equal amounts of cis9,trans11–18:2 and trans10,cis12–18:2; the two most common isoforms of CLA. Milk was collected by complete breast expression on the last day (day 5) of each intervention period and analyzed for macronutrient and fatty acid composition. On day 4 of each intervention period, infant milk consumption was estimated by 24 h weighing of the infant. Washout periods were 9 days in length. We observed a dose-dependent increase in the concentrations of cis9,trans11–18:2 and trans10,cis12–18:2 in the milk fat. However, we detected neither a change in overall macronutrient composition nor infant milk consumption. These data do not support those obtained from animal models or our previous human work suggesting that consumption of CLA mixtures necessarily reduces milk fat. It is possible that either (1) the interpretation of our previously published data should be reevaluated, and/or (2) there are important intra- and inter-species differences in this regard.     

Human milk in disease: Lipid Composition

Differences in the lipid composition of human milk have been described in maternal diseases known to affect fat metabolism. Diseases such as diabetes, cystic fibrosis, hypobetalipoproteinemia and Type I hyperlipoproteinemia affect the quantity and quality of human milk fat. Increased fatty acid chain elongation and changes in desaturation (especially Δ6 desaturase), as well as changes in lipid class composition, have been shown in diabetes and cystic fibrosis, whereas compensatory increases in medium-chain fatty acids have been described in hypobetalipoproteinemia and Type I hyperlipoproteinemia. It is important to realize that these observations were made either on single women or on very small groups of women. In infant diseases, such as breast milk jaundice and ectopic eczema, changes in polyunsaturated fatty acids in maternal milk have been described. Based on a paper presented at the Symposium on Milk Lipids held at the AOCS Annual Meeting, Baltimore, MD, April 1990.     

Vergleich des Triglyceridmusters von Humanmilch und Säuglingsnahrungen

Comparison between the triglyceride Patterns of Human Milk and Infant Milk Formulae Human milk contains less triglycerides with saturated fatty acids than the three infant milk formulae analysed but has a higher content of triglycerides with two unsaturated fatty acids. The patterns of the major triglycerides are compared. In human milk, palmitic acid is preferably located in position 2 favourable to digestability, whereas in the infant milk formulae it is mostly distributed over positions 1 and 3.     

Large-scale in vivo synthesis of the carbohydrate moieties of gangliosides GM1 and GM2 by metabolically engineered Escherichia coli

Two metabolically engineered Escherichia coli strains have been constructed to produce the carbohydrate moieties of gangliosides GM2 (GalNAcbeta-4(NeuAcalpha-3)Galbeta-4Glc; Gal = galactose, Glc = glucose, Ac = acetyl) and GM1 (Galbeta-3GalNAcbeta-4(NeuAcalpha-3)Galbeta-4Glc. The GM2 oligosaccharide-producing strain TA02 was devoid of both beta-galactosidase and sialic acid aldolase activities and overexpressed the genes for CMP-NeuAc synthase (CMP = cytidine monophosphate), alpha-2,3-sialyltransferase, UDP-GlcNAc (UDP = uridine diphosphate) C4 epimerase, and beta-1,4-GalNAc transferase. When this strain was cultivated on glycerol, exogenously added lactose and sialic acid were shown to be actively internalized into the cytoplasm and converted into GM2 oligosaccharide. The in vivo synthesis of GM1 oligosaccharide was achieved by taking a similar approach but using strain TA05, which additionally overexpressed the gene for beta-1,3-galactosyltransferase. In high-cell-density cultures, the production yields for the GM2 and GM1 oligosaccharides were 1.25 g L(-1) and 0.89 g L(-1), respectively.     

Melatonin in Early Nutrition: Long-Term Effects on Cardiovascular System

Breastfeeding protects against adverse cardiovascular outcomes in the long term. Melatonin is an active molecule that is present in the breast milk produced at night beginning in the first stages of lactation. This indoleamine appears to be a relevant contributor to the benefits of breast milk because it can affect infant health in several ways. The melatonin concentration in breast milk varies in a circadian pattern, making breast milk a chrononutrient. The consumption of melatonin can induce the first circadian stimulation in the infant’s body at an age when his/her own circadian machinery is not functioning yet. This molecule is also a powerful antioxidant with the ability to act on infant cells directly as a scavenger and indirectly by lowering oxidant molecule production and enhancing the antioxidant capacity of the body. Melatonin also participates in regulating inflammation. Furthermore, melatonin can participate in shaping the gut microbiota composition, richness, and variation over time, also modulating which molecules are absorbed by the host. In all these ways, melatonin from breast milk influences weight gain in infants, limiting the development of obesity and comorbidities in the long term, and it can help shape the ideal cellular environment for the development of the infant’s cardiovascular system.     

The DINO trial – challenges for translation into clinical practice

Infants born prematurely require higher amounts of dietary docosahexaenoic acid (DHA) than can be supplied through current strategies. Increasing the DHA content of breast milk and formula while achievable may not be the most efficacious approach. A new approach is required that enables direct delivery to the infant enabling maximum dose to be achieved as early as possible.     

Infant formulas and the use of vegetable protein

Soy-based formulas and meat-based formulas are used as successful replacements for milk in the nutritional management of infants who are, or are suspected to be allergic to milk. Used most widely are soy-based formulas which eliminate the symptoms and ensure normal growth and well being of the infant. Soy-based formulas are made in both powdered and liquid forms, and the trend during the past decade has been to use a soy protein isolate to reduce or eliminate the presence of carbohydrates which cause flatulence and abnormal stooling. Details of the nutritional composition of soy-based infant formulas are discussed together with the selection and processing of protein in order to minimize the presence of antinutritional factors.     

Gangliosides of cultured cells of a rat mammary carcinoma cell line

Ganglioside content of rat mammary carcinoma-derived cells grown in layers in vitro was nearly as high as that of apical plasma membrane-derived milk fat globule membrane and nearly four times higher than the content of normal, lactating mammary tissue on a protein basis. The major ganglioside of these carcinoma-derived cells was identified as GDla (sialic acid-Gal-GalNAc-(sialic acid)-Gal-Glc-Cer. Relative to carcinoma-derived cells, rat mammary tissue and milk fat globule membrane had more complex ganglioside patterns but appeared to lack substantial quantities of GDla.     

Trans,n-3, and n-6 fatty acids in canadian human milk

The presence oftrans fatty acids in human milk may be a concern because of their possible adverse nutritional and physiological effects on the recipient infant. The mother's diet is the source of human milktrans fatty acids, and since these fatty acids are prevalent in many common foods of the Canadian diet, thetrans fatty acid content and the fatty acid composition of Canadian human milk were measured by gas-liquid chromatography coupled with silver nitrate-thin layer chromatography. In samples obtained from 198 lactating mothers across Canada, the average percentage of totaltrans (sum oft18∶1,t18∶2, andt18∶3) was 7.2% of breast milk fatty acids with a range of 0.1–17.2%. Analysis oft18∶1 isomer distribution indicated that partially hydrogenated vegetable oils are the major source of thesetrans fatty acids in human milk, whereas contribution from dairy products appeared to be relatively minor. Linoleci and α-linolenic acid levels were inversely related to the totaltrans fatty acids, indicating that the elevation oftrans fatty acids in Canadian human milk is at the expense of n-3 and n-6 essential fatty acids. Levels of arachidonic and docosahexaenoic acids did not correlate with their parent fatty acids, indicating that it might be difficult to elevate the levels of n-6 and n-3 C_20–22 polyunsaturated fatty acids in breast milk by increasing levels of linoleic and α-linolenic acids in the mother's diet.     

Exploring the Role of Different Neonatal Nutrition Regimens during the First Week of Life by Urinary GC-MS Metabolomics

In this study, a gas-chromatography mass spectrometry (GC-MS) metabolomics study was applied to examine urine metabolite profiles of different classes of neonates under different nutrition regimens. The study population included 35 neonates, exclusively either breastfed or formula milk fed, in a seven-day timeframe. Urine samples were collected from intrauterine growth restriction (IUGR), large for gestational age (LGA), and appropriate gestational age (AGA) neonates. At birth, IUGR and LGA neonates showed similarities in their urine metabolite profiles that differed from AGA. When neonates started milk feeding, their metabolite excretion profile was strongly characterized by the different diet regimens. After three days of formula milk nutrition, urine had higher levels of glucose, galactose, glycine and myo-inositol, while up-regulated aconitic acid, aminomalonic acid and adipic acid were found in breast milk fed neonates. At seven days, neonates fed with formula milk shared higher levels of pseudouridine with IUGR and LGA at birth. Breastfed neonates shared up-regulated pyroglutamic acid, citric acid, and homoserine, with AGA at birth. The role of most important metabolites is herein discussed.     

Process optimization of simultaneous enzymatic production of 1,3-dioleoyl-2-palmitoylglycerol and 1-oleoyl-2-palmitoyl-3-linoleoylglycerol

1,3-dioleoyl-2-palmitoylglycerol (OPO), a structured triacylglycerols (TAG) containing palmitic acid at sn-2 position, is widely used as breast milk fat substitutes in infant formula. Interestingly, the main component of breast milk fat in Western countries is OPO while Chinese breast milk contains both 1-oleoyl-2-palmitoyl-3-linoleoylglycerol (OPL) and OPO. Thus, OPL is additionally essential for Chinese infants. In the present study, we developed a process for simultaneous production of OPL and OPO. According to the fatty acid (FA) composition of Chinese breast milk, soy oil, palm kernel stearin, and palm stearin were selected as the base oils for random interesterification, and the appropriate ratio was determined as 2:3:15. Then interesterification at sn-1,3 position was conducted, four reaction parameters were examined, where 1.6 molar ratio of oleic acid to linoleic acid (OLA/LNA), 10% enzyme quantity, TAG to FA ratio of 1:8 and reaction temperature of 56°C were ultimately established as the optimal conditions to achieve the target of the present study, and the OPO and OPL contents in the final products were 23.1% and 26.2%, respectively. Finally, molecular distillation was applied to purify above achieved breast milk fat substrates. Higher oil dropping speed, oil temperature, and agitation speed were favorable conditions, and the lowest acid value that achieved in the present study was 3.46 mgKOH/g. Overall, we established an efficient preliminary process for the simultaneous production of OPO and OPL, which could provide an important reference for commercial production of breast milk fat substrates for Chinese infants.     

Isomeric fatty acids: Evaluating status and implications for maternal and child health

“Isomeric fatty acids” is a term that refers to the trans- and positional isomers formed during hydrogenation of naturally occurring oils. The purposes of this paper are as follows: (i) to summarize potential exposure of infants to isomeric fatty acids by reviewing estimates of isomeric fatty acids in the maternal diet, in human milk, and in infant formula/infant foods, and (ii) to evaluate the evidence for adverse effects of isomeric fatty acids on infant development with respect to growth and essential fatty acid status. Estimates of the intake of trans-fatty acids vary widely both within and across populations. Current estimates of trans-fatty acids in the North American population are 4–11% of total fatty acids or 3–13 g/(person·d), whereas in Mediterranean countries in which olive oil is the primary fat and in Far Eastern countries in which little commercially hydrogenated fat is consumed, per capita consumption of trans-fatty acids is <1–2 g/d. The trans-fatty acid content of human milk reflects the cross-cultural variation in the maternal diet, with trans-fatty acids in human milk samples ranging from 6 to 7% in North America to <0.5% in Hong Kong. Trans-fatty acids are transferred from the maternal diet through the placenta to the developing fetus or through milk to the breast-fed infant. In some studies, plasma trans-fatty acids are inversely related to birth weight and head circumference. The hypothesis that dietary trans-fatty acids could inhibit biosynthesis of long-chain polyunsaturated fatty acids with 20 and 22 carbon atoms and thus affect infant development is supported by studies demonstrating an inverse correlation of plasma trans-fatty acids with n−3 and n−6 long-chain polyunsaturated fatty acids in infants. However, no such relationship has been observed in human milk. A definitive answer concerning a potentially adverse effect of dietary trans-fatty acids on infant development awaits future studies.     

Human Milk Fat Substitute from Butterfat: Production by Enzymatic Interesterification and Evaluation of Oxidative Stability

Recent data have suggested that the fatty acid composition and molecular structure of fats in infant formulas should be as similar to human milk fat as possible to obtain optimal fat and calcium absorption from the infant formula. This work investigated the possibilities of using enzyme technology and butterfat as a material to produce a fat similar to human milk fat with respect to the above parameters. Moreover, the oxidative stability of the enzyme modified human milk fat substitute (HMFS) was compared to the fat blend used for the production of HMFS. Using a combination of enzyme technology, fractionation and batch deodorization and with butterfat in combination with soybean oil and rapeseed oil as raw materials it was possible to produce HMFS with a molecular structure and fatty acid composition that was very similar to that of human milk fat. The oxidative stability of the HMFS oil was lower than that of the reference oil with the same fatty acid composition. However, oxidation did not lead to a severe increase in rancidity scores during storage. Rather, the panel gave high intensity scores for other off-flavors such as burnt and bitter. Further optimization of the deodorization process is therefore necessary to remove these off-flavors.