Quantification of Individual Oligosaccharide Compounds from Human Milk Using High-pH Anion-Exchange Chromatography

Thurl, Stephan; Müller-Werner, Beate; Sawatzki, G.

doi:10.1006/abio.1996.0113

Cited by 157 publications

(136 citation statements)

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“…The analytical methods that are capable of separating and characterizing the various sugar compositions and structures of oligosaccharides in human milk include high-performance liquid chromatography (HPLC), high pH anion exchange chromatography (HPAEC), capillary electrophoresis and various mass spectrometry platforms (MS) [13][14][15][16][17][18][19][20][21]. These methods as currently used are technically cumbersome, incapable of producing large quantities of highly purified isolated molecules and, as a result, there is little information on many of the basic biological properties of this class of molecule.…”

Section: Milk Oligosaccharidesmentioning

confidence: 99%

Human Milk Oligosaccharides: Evolution, Structures and Bioselectivity as Substrates for Intestinal Bacteria

German

Freeman

Lebrilla

et al. 2008

Nestlé Nutrition Workshop Series: Pediatric Program

197

170

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Human milk contains a high concentration of diverse soluble oligosaccharides, carbohydrate polymers formed from a small number of monosaccharides. Novel methods combining liquid chromatography with high resolution mass spectrometry have identified approximately 200 unique oligosaccharides structures varying from 3 to 22 sugars. The increasing complexity of oligosaccharides follows the general pattern of mammalian evolution though the concentration and diversity of these structures in homo sapiens are strikingly. There is also diversity among human mothers in oligosaccharides. Milks from randomly selected mothers contain as few as 23 and as many as 130 different oligosaccharides. The functional implications of this diversity are not known. Despite the role of milk to serve as a sole nutrient source for mammalian infants, the oligosaccharides in milk are not digestible by human infants. This apparent paradox raises questions about the functions of these oligosaccharides and how their diverse molecular structures affect their functions. The nutritional function most attributed to milk oligosaccharides is to serve as prebiotics -a form of indigestible carbohydrate that is selectively fermented by desirable gut microflora. This function was tested by purifying human milk oligosaccharides and providing these as the sole carbon source to various intestinal bacteria. Indeed, the selectively of providing the complex mixture of oligosaccharides pooled from human milk samples is remarkable. Among a variety of Bifidobacteria tested only Bifidobacteria longum biovar infantis was able to grow extensively on human milk oligosaccharides as sole carbon source. The genomic sequence of this strain revealed approximately 700 genes that are unique to infantis, including a variety of co-regulated glycosidases, relative to other Bifidobacteria, implying a co-evolution of human milk oligosaccharides and the genetic capability of select intestinal bacteria to utilize them. The goal of ongoing research is to assign specific functions to the combined oligosaccharide-bacteria-host interactions that emerged from this evolutionary pressure.

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Section: Milk Oligosaccharidesmentioning

confidence: 99%

Human Milk Oligosaccharides: Evolution, Structures and Bioselectivity as Substrates for Intestinal Bacteria

German

Freeman

Lebrilla

et al. 2008

Nestlé Nutrition Workshop Series: Pediatric Program

197

170

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“…As revealed by MALDI-MS [51,52], human milk contains, besides some major components, large-size oligosaccharides up to 8000 Da of so far unknown structure. The combination of high pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) [53,54] and MALDI-MS proved to be a powerful tool for the analysis of human milk oligosaccharides with high sensitivity and no need of derivatization [52].…”

Section: Mass Spectrometry Of Human Milk Oligosaccharidesmentioning

confidence: 99%

“…The separation of oligosaccharides from pooled human milk was performed using the following techniques: pasteurization, centrifugation, precipitation of proteins, preparative anion-exchange chromatography, HPLC chromatography, and GPC prefractionation; a more detailed description is given in [7,[52][53][54].…”

Section: Samplesmentioning

confidence: 99%

Structural analysis of underivatized neutral human milk oligosaccharides in the negative ion mode by nano-electrospray MSⁿ (Part 1: Methodology)

Pfenninger

Karas

Finke³

et al. 2002

J. Am. Soc. Mass Spectrom.

155

133

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Underivatized neutral oligosaccharides from human milk were analyzed by nano-electrospray ionization (ESI) using a quadrupole ion trap mass spectrometer (QIT-MS) in the negative-ion mode. Under these conditions neutral oligosaccharides are observed as deprotonated molecules [M Ϫ H] Ϫ with high intensity. CID-experiments of these species with the charge localized at the reducing end lead to C-type fragment ions forming a "new" reducing end. Fragmentations are accompanied by cross-ring cleavages that yield information about linkages of internal monosaccharides. Several isomeric compounds with distinct structural features, such as different glycosidic linkages, fucosylation and branching sites were investigated. The rules governing the fragmentation behavior of this class of oligosaccharides were elucidated and tested for a representative number of certain isomeric glycoforms using the MS/MS and MS n capabilities of the QIT. On the basis of the specific fragmentation behavior of deprotonated molecules, the position of fucoses and the linkage type (Gal ␤133 GlcNAc or Gal ␤134 GlcNAc) could be determined and linear and branched could be differentiated. Rules could be established which can be applied in further investigations of these types of oligosaccharides even from heterogenous mixtures. . It creates new demands for analytical tools for structure elucidation of complex oligosaccharides comprising composition, sequence, branching, and linkage analysis, including anomericity and finally also ring sizes and absolute configuration, i.e., identity of the subunits. Mass spectrometry (MS) offers the possibility of structural investigations of oligosaccharides; this has been demonstrated using fast atom bombardment (FAB-MS) [2,3]. Electrospray (ESI) MS and matrix-assisted laser desorption ionization (MALDI) MS have been applied to the investigation of carbohydrates of biological origin [4,5]. Their ability to deliver structural information on different levels depends on the mass analyzer coupled to the ion source. In general, mass spectrometry provides the possibility of structural elucidation based on characteristic fragmentations of the molecules under investigation. A nomenclature for the possible fragment ions of oligosaccharides has been proposed by Domon and Costello [6], i.e., B and C for fragment ions containing the nonreducing side, Y and Z for those containing the reducing sugar unit, as well as A and X type fragment ions for those arising from cross-ring cleavages.In MALDI-MS neutral oligosaccharides are usually observed as singly-cationized (typically sodiated) species [7], which offers a simple means for screening complex mixtures. As MALDI is mostly coupled to a time-of-flight (TOF) mass analyzer, options for structural analysis are restricted to metastable fragment-ion analysis post-source decay (PSD) [8]. Some successful attempts for structure elucidation have been made for underivatized and derivatized oligosaccharides by MALDI/PSD analysis [9 -15]. However, fragmentation is poorly controllable, but some...

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“…The separation of oligosaccharides from pooled human milk was performed as described in [4,[17][18][19]; in this study, GPC subfractions of neutral oligosaccharides were analyzed. For mass analysis the lyophilized GPCsubfractions were dissolved in deionized water to a total concentration of 1 g/L; ammonium acetate (10 Ϫ3 M) was used as additive (3:1,vol:vol).…”

Section: Sample Preparationmentioning

confidence: 99%

Structural analysis of underivatized neutral human milk oligosaccharides in the negative ion mode by nano-electrospray MSⁿ (Part 2: Application to isomeric mixtures)

Pfenninger

Karas

Finke³

et al. 2002

J. Am. Soc. Mass Spectrom.

113

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A complex mixture of isomeric neutral oligosaccharides from pooled human milk was analyzed by nano-electrospray ionization (ESI) in a quadrupole ion trap mass spectrometer (QIT-MS) in the negative ion mode. Since deprotonated molecules of neutral oligosaccharides follow distinct fragmentation rules, which have been elucidated by using model compounds (see [1]), spectra obtained from consecutive CID experiments allowed the differentiation of isomers out of this highly complex mixture. With this method new human milk oligosaccharides of previously unknown isomeric structures have been identified, e.g., the occurence of three isomeric fucosylated lacto-N-hexaoses could be determined precisely, which have not been described before: . Also, free oligosaccharides, which are present either extracellular or in cell matrices, are believed to have other than nutritional function. As the (bio)chemical basis of many life processes is known, both recognition of diseases and the development of medical treatment becomes possible. Also preventional health treatment gains importance; especially nutrition is known to be an essential factor. This leads to the development of nutritional additives with healing and precautional character (functional food).As revealed by MALDI/MS human milk contains neutral as well as acidic oligosaccharides of high molecular weight [3,4], of which the biological function is yet not known, but there is some evidence that these compounds have essential biological functions [5,6]. It is hypothesized that due to their structural similarity to the sugars in N-and O-glycans and their resistance against digestion by glycosidases specific for N-and Oglycans, human milk oligosaccharides are able to reach the colon. There they may be potential, water-soluble receptor analogues which prevent interactions of pathogenic bacteria, viruses, fungi, or effects of toxins. They also may have benificial effects for the development of a specific intestinal flora of breast-fead infants. As they are potential nonimmunological factors, human milk oligosaccharides are under discussion as supplements for infant formula. Therefore, knowledge of their specific biological activity and the crucial concentration is necessary.This demands analytical tools that provide structural elucidation of bioactive compounds and their active concentration. Today, there is no universal method for the characterization of oligosaccharides. Traditional methods for structural analysis of oligosaccharides are time and cost consuming. Even though NMR spectroscopy as well as X-ray crystallography have been successfully applied [7,8], both methods need high amounts of sample in high purity. Mass spectrometry is a useful and sensitive method to investigate samples of low absolute amount and concentration or in complex mixtures. The useful combination of classical enzymatic approach and mass spectrometry is not only time consuming and costly, it is also limited to samples, that

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Quantification of Individual Oligosaccharide Compounds from Human Milk Using High-pH Anion-Exchange Chromatography

Cited by 157 publications

References 1 publication

Human Milk Oligosaccharides: Evolution, Structures and Bioselectivity as Substrates for Intestinal Bacteria

Human Milk Oligosaccharides: Evolution, Structures and Bioselectivity as Substrates for Intestinal Bacteria

Structural analysis of underivatized neutral human milk oligosaccharides in the negative ion mode by nano-electrospray MSⁿ (Part 1: Methodology)

Structural analysis of underivatized neutral human milk oligosaccharides in the negative ion mode by nano-electrospray MSⁿ (Part 2: Application to isomeric mixtures)

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Quantification of Individual Oligosaccharide Compounds from Human Milk Using High-pH Anion-Exchange Chromatography

Cited by 157 publications

References 1 publication

Human Milk Oligosaccharides: Evolution, Structures and Bioselectivity as Substrates for Intestinal Bacteria

Human Milk Oligosaccharides: Evolution, Structures and Bioselectivity as Substrates for Intestinal Bacteria

Structural analysis of underivatized neutral human milk oligosaccharides in the negative ion mode by nano-electrospray MSn (Part 1: Methodology)

Structural analysis of underivatized neutral human milk oligosaccharides in the negative ion mode by nano-electrospray MSn (Part 2: Application to isomeric mixtures)

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Structural analysis of underivatized neutral human milk oligosaccharides in the negative ion mode by nano-electrospray MSⁿ (Part 1: Methodology)

Structural analysis of underivatized neutral human milk oligosaccharides in the negative ion mode by nano-electrospray MSⁿ (Part 2: Application to isomeric mixtures)