Milk sugars beyond lactose : metabolic fate of neutral milk oligosaccharides in infants

Free oligosaccharides in human milk (HMO) are complex carbohydrates structurally based on lactose and present at concentrations of 5–20 g/L. They are considered to be resistant to digestion in the breastfed infant’s stomach and small intestine, and to serve as energy source selectively for beneficial microbiota in the bowel. Apart from this prebiotic effect, HMO have been found to exert various biological activities in numerous in vitro studies, that indicate not only local functionality in the gut, but also, e.g. immunomodulatory or anti-infective, effects within the body. However, a direct link between structure and function in vivo has not yet been provided. This, in turn, is due to the enormous structural diversity of HMO: More than 150 different structures have been characterized to date. In this work, the metabolic pathways and excretion profiles of HMO were investigated in vivo to extend our knowledge on the sites of HMO utilization or modification and thereby to provide hints on the structure-function relationship of HMO in the infant. Therefore, a platform based on solid phase extraction and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for a high throughput-profiling of oligosaccharides from human milk, infants’ urine and feces was established, complemented by isomer separation via liquid chromatography. It was used to follow the time course and possible metabolites of 13C-enriched and non-enriched oligosaccharides in milk and infant urine from ten mother-infant pairs during 36 hours after the application of 13C-galactose to the mothers. Intestinal absorption and subsequent renal excretion of intact structures, in particular (fucosylated) hexaoses, could be deduced for all infants. However, in some infants, excretion was delayed, pointing at a longer gut passage or systemic circulation of those certain structures. Moreover, structure-specific modifications and utilization in the upper parts of the gut, where absorption is thought to take place, were hypothesized. The overall oligosaccharide profiles in the feces of breastfed infants could be categorized into three groups, i.e. i) high diversity with many HMO-like structures, ii) only one or a few oligosaccharides with rather low signal intensity, and iii) no HMO. The patterns showed some association with the infants’ age. However, further research is needed to investigate the underlying causes, e.g. gut maturity or stool frequency. Novel metabolites were identified in both, infant urine and feces. They could be characterized as acetylated HMO or HMO degradation/modification products, generated by the infants or by their gut microbiota, such as secretor- or Lewis-specific HMO in the feces/urine of infants fed nonsecretor or Lewis-negative milk. Lacto-N-tetraose as a major oligosaccharide in milk was significantly reduced especially in fecal samples. Moreover, the secretor-specific structure lacto-N-fucopentaose I, which is highly abundant in secretor milk, was not detected in the urine of the infants fed Lewis b secretor milk, which indicates a selective utilization of this specific structure.Humanmilch enthält eine Vielzahl an komplexen Kohlenhydraten, die sich biosynthetisch von dem Milchzucker Laktose ableiten. Ihre Konzentrationen betragen mit ca. 5–20 Gramm pro Liter etwa ein Zehntel des Gehalts von Laktose. Bisher wurden zahlreiche biologische Effekte von Humanmilcholigosacchariden (HMO) anhand von Ergebnissen aus in vitro Studien beschrieben. Sie sollen u.a. präbiotisch und antiinfektiös im Darm des gestillten Säuglings wirken. Darüber hinaus könnten sie auch immunmodulierende und andere positive Eigenschaften im Körper haben, sofern die aktiven Substanzen im Dünndarm absorbiert werden. Um den endgültigen Beweis für die Wirksamkeit der einzelnen Substanzen beim Säugling in vivo zu erbringen, wären Interventionsstudien mit Einzelsubstanzen notwendig. Diese sind aufgrund mangelnder Verfügbarkeit synthetischer HMO bislang nicht möglich. Daher wurden in der vorliegenden Arbeit der Metabolismus und die möglichen Ausscheidungswege von HMO direkt beim gestillten Säugling untersucht, um so Hinweise auf deren Struktur-Funktionsbeziehungen in vivo zu erhalten. Dafür wurde eine analytische Plattform entwickelt, die eine effiziente und zuverlässige Identifizierung von Oligosacchariden aus Muttermilch, Säuglingsurin und –fäzes ermöglicht. In Kapitel 1 der vorliegenden Arbeit werden die aktuellen Kenntnisse zum Metabolismus und der Funktion von HMO sowie die gängigsten Methoden der Glykananalytik anhand von Literatur vorgestellt. Kapitel 2 verdeutlicht die große Relevanz der modernen Glykananalytik für die Erforschung der Struktur-Funktionsbeziehungen von HMO mit Fokus auf spezifische HMO-Strukturen mit Lewis- oder Sekretorepitopen (genetisch determinierte Blutgruppenantigene in Humanmilch). Kapitel 3 beschreibt die praktischen Studien zum Zeitverlauf der HMO-Ausscheidung im Säuglingsurin 36 Stunden nach Einzelgabe von 13C-Galaktose an die Mütter. Mittels Matrix-unterstützter Laser Desorption/Ionizations-Massenspektrometrie mit Flugzeitanalysator (MALDI-TOF-MS) wurden bei allen zehn Säuglingen intakte, auch sehr komplexe HMO detektiert. Allerdings war die Exkretion bestimmter Strukturen bei manchen Kindern verzögert, was auf eine individuell längere Darmpassage oder Blutzirkulation dieser hinweist. Des Weiteren gab es Hinweise auf eine unerwartet frühe Metabolisierung bestimmter Strukturen in oberen Darmabschnitten. Die möglichen Metaboliten nach ihrem Abbau oder Modifikation und/oder Aufnahme im Darm wurden bei derselben Probandengruppe mittels MALDI-TOF-MS und Flüssigchromatographie untersucht (Kapitel 4). Diese konnten mittels Tandem-MS als acetylierte HMO oder verschiedene HMO-Abbauprodukte charakterisiert werden, zusätzlich zu HMO-Strukturen, die offenbar erst nach der Aufnahme von den Enzymen des Kindes mit Lewis- und/oder Sekretorepitopen versehen worden waren. Einige Hinweise auf strukturspezifische Utilisation im Darm oder Organismus des Säuglings wurden darüber hinaus erlangt, beispielsweise aufgrund stark verminderter Ausscheidung von Lakto-N-tetraose im Stuhl oder Lacto-N-fukopentaose I im Urin. Die Oligosaccharidmuster von weiteren 24 Säuglingen wurden ebenfalls mittels MALDI-TOF-MS untersucht (Kapitel 5). Dabei wurden drei Untergruppen anhand der HMO-Muster im Stuhl der gestillten Säuglinge im Alter von sechs Wochen beobachtet: i) hohe Diversität an Oligosaccharidstrukturen mit Dominanz von intakten HMO, ii) wenige Oligosaccharide mit eher niedriger Signalintensität und iii) keine HMO oder HMO-Metaboliten. Im Alter von sechs Monaten wurden bei den gestillten Säuglingen keine oder sehr wenige HMO(Metaboliten) detektiert. Des Weiteren konnten bei den formelernährten und bei zwei von drei gemischt ernährten Säuglingen keine Oligosaccharide im Stuhl detektiert werden. In Kapitel 6 werden die neuen Erkenntnisse auf der Basis vorhandener Literatur abschließend diskutiert und es werden Perspektiven für künftige Humanstudien über HMO aufgezeigt

Comparative Glycomics of Immunoglobulin A and G From Saliva and Plasma Reveals Biomarker Potential

The N-glycosylation of immunoglobulin (Ig) G, the major antibody in the circulation of human adults, is well known for its influence on antibody effector functions and its alterations with various diseases. In contrast, knowledge on the role of glycans attached to IgA, which is a key immune defense agent in secretions, is very scarce. In this study we aimed to characterize the glycosylation of salivary (secretory) IgA, including the IgA joining chain (JC), and secretory component (SC) and to compare IgA and IgG glycosylation between human plasma and saliva samples to gain a first insight into oral cavity-specific antibody glycosylation. Plasma and whole saliva were collected from 19 healthy volunteers within a 2-h time window. IgG and IgA were affinity-purified from the two biofluids, followed by tryptic digestion and nanoLC-ESI-QTOF-MS(/MS) analysis. Saliva-derived IgG exhibited a slightly lower galactosylation and sialylation as compared to plasma-derived IgG. Glycosylation of IgA1, IgA2, and the JC showed substantial differences between the biofluids, with salivary proteins exhibiting a higher bisection, and lower galactosylation and sialylation as compared to plasma-derived IgA and JC. Additionally, all seven N-glycosylation sites, characterized on the SC of secretory IgA in saliva, carried highly fucosylated and fully galactosylated diantennary N-glycans. This study lays the basis for future research into the functional role of salivary Ig glycosylation as well as its biomarker potential

IgG N-glycans are associated with prevalent and incident complications of type 2 diabetes

Aims/Hypothesis:Inflammation is important in the development of type 2 diabetes complications. The N-glycosylation of IgG influences its role in inflammation. To date, the association of plasma IgG N-glycosylation with type 2 diabetes complications has not been extensively investigated. We hypothesised that N-glycosylation of IgG may be related to the development of complications of type 2 diabetes. Methods: In three independent type 2 diabetes cohorts, plasma IgG N-glycosylation was measured using ultra performance liquid chromatography (DiaGene n = 1815, GenodiabMar n = 640) and mass spectrometry (Hoorn Diabetes Care Study n = 1266). We investigated the associations of IgG N-glycosylation (fucosylation, galactosylation, sialylation and bisection) with incident and prevalent nephropathy, retinopathy and macrovascular disease using Cox- and logistic regression, followed by meta-analyses. The models were adjusted for age and sex and additionally for clinical risk factors. Results: IgG galactosylation was negatively associated with prevalent and incident nephropathy and macrovascular disease after adjustment for clinical risk factors. Sialylation was negatively associated with incident diabetic nephropathy after adjustment for clinical risk factors. For incident retinopathy, similar associations were found for galactosylation, adjusted for age and sex. Conclusions: We showed that IgG N-glycosylation, particularly galactosylation and to a lesser extent sialylation, is associated with a higher prevalence and future development of macro- and microvascular complications of diabetes. These findings indicate the predictive potential of IgG N-glycosylation in diabetes complications and should be analysed further in additional large cohorts to obtain the power to solidify these conclusions.</p

Large-Scale Analysis of Apolipoprotein CIII Glycosylation by Ultrahigh Resolution Mass Spectrometry

Apolipoprotein-CIII (apo-CIII) is a glycoprotein involved in lipid metabolism and its levels are associated with cardiovascular disease risk. Apo-CIII sialylation is associated with improved plasma triglyceride levels and its glycosylation may have an effect on the clearance of triglyceride-rich lipoproteins by directing these particles to different metabolic pathways. Large-scale sample cohort studies are required to fully elucidate the role of apo-CIII glycosylation in lipid metabolism and associated cardiovascular disease. In this study, we revisited a high-throughput workflow for the analysis of intact apo-CIII by ultrahigh-resolution MALDI FT-ICR MS. The workflow includes a chemical oxidation step to reduce methionine oxidation heterogeneity and spectrum complexity. Sinapinic acid matrix was used to minimize the loss of sialic acids upon MALDI. MassyTools software was used to standardize and automate MS data processing and quality control. This method was applied on 771 plasma samples from individuals without diabetes allowing for an evaluation of the expression levels of apo-CIII glycoforms against a panel of lipid biomarkers demonstrating the validity of the method. Our study supports the hypothesis that triglyceride clearance may be regulated, or at least strongly influenced by apo-CIII sialylation. Interestingly, the association of apo-CIII glycoforms with triglyceride levels was found to be largely independent of body mass index. Due to its precision and throughput, the new workflow will allow studying the role of apo-CIII in the regulation of lipid metabolism in various disease settings

Effluent and serum protein N-glycosylation is associated with inflammation and peritoneal membrane transport characteristics in peritoneal dialysis patients

Abstract Mass spectrometric glycomics was used as an innovative approach to identify biomarkers in serum and dialysate samples from peritoneal dialysis (PD) patients. PD is a life-saving treatment worldwide applied in more than 100,000 patients suffering from chronic kidney disease. PD treatment uses the peritoneum as a natural membrane to exchange waste products from blood to a glucose-based solution. Daily exposure of the peritoneal membrane to these solutions may cause complications such as peritonitis, fibrosis and inflammation which, in the long term, lead to the failure of the treatment. It has been shown in the last years that protein N-glycosylation is related to inflammatory and fibrotic processes. Here, by using a recently developed MALDI-TOF-MS method with linkage-specific sialic acid derivatisation, we showed that alpha2,6-sialylation, especially in triantennary N-glycans from peritoneal effluents, is associated with critical clinical outcomes in a prospective cohort of 94 PD patients. Moreover, we found an association between the levels of presumably immunoglobulin-G-related glycans as well as galactosylation of diantennary glycans with PD-related complications such as peritonitis and loss of peritoneal mesothelial cell mass. The observed glycomic changes point to changes in protein abundance and protein-specific glycosylation, representing candidate functional biomarkers of PD and associated complications

Total serum N-glycans mark visceral leishmaniasis in human infections with Leishmania infantum

Summary: Visceral leishmaniasis (VL) is a clinical form of leishmaniasis with high mortality rates when not treated. Diagnosis suffers from invasive techniques and sub-optimal sensitivities. The current (affordable) treatment with pentavalent antimony as advised by the WHO is possibly harmful to the patient. There is need for an improved diagnosis to prevent possibly unnecessary treatment. N-glycan analysis may aid in diagnosis. We evaluated the N-glycan profiles from active VL, asymptomatic infections (ASYMP) and controls from non-endemic (NC) and endemic (EC) areas. Active VL has a distinct N-glycome profile that associates with disease severity. Our study suggests that the observed glycan signatures could be a valuable additive to diagnosis and assist in identifying possible markers of disease and understanding the pathogenesis of VL. Further studies are warranted to assess a possible future role of blood glycome analysis in active VL diagnosis and should aim at disease specificity

Metformin and statin use associate with plasma protein N -glycosylation in people with type 2 diabetes

Introduction Recent studies revealed N-glycosylation signatures of type 2 diabetes, inflammation and cardiovascular risk factors. Most people with diabetes use medication to reduce cardiovascular risk. The association of these medications with the plasma N-glycome is largely unknown. We investigated the associations of metformin, statin, ACE inhibitor/angiotensin II receptor blocker (ARB), sulfonylurea (SU) derivatives and insulin use with the total plasma N-glycome in type 2 diabetes. Research design and methods After enzymatic release from glycoproteins, N-glycans were measured by matrix-assisted laser desorption/ionization mass spectrometry in the DiaGene (n=1815) and Hoorn Diabetes Care System (n=1518) cohorts. Multiple linear regression was used to investigate associations with medication, adjusted for clinical characteristics. Results were meta-analyzed and corrected for multiple comparisons. Results Metformin and statins were associated with decreased fucosylation and increased galactosylation and sialylation in glycans unrelated to immunoglobulin G. Bisection was increased within diantennary fucosylated non-sialylated glycans, but decreased within diantennary fucosylated sialylated glycans. Only few glycans were associated with ACE inhibitor/ARBs, while none associated with insulin and SU derivative use. Conclusions We conclude that metformin and statins associate with a total plasma N-glycome signature in type 2 diabetes. Further studies are needed to determine the causality of these relations, and future N-glycomic research should consider medication a potential confounder

Simultaneous Immunoglobulin A and G Glycopeptide Profiling for High-Throughput Applications

Immunoglobulin (Ig) glycosylation is recognized for its influence on Ig turnover and effector functions. However, the large-scale profiling of Ig glycosylation in a biomedical setting is challenged by the existence of different Ig isotypes and subclasses, their varying serum concentrations, and the presence of multiple glycosylation sites per Ig. Here, a high-throughput nanoliquid chromatography (LC)- mass spectrometry (MS)-based method for simultaneous analysis of IgG and IgA glycopeptides was developed and applied on a serum sample set from 185 healthy donors. Sample preparation from minute amounts of serum was performed in 96-well plate format. Prior to trypsin digestion, IgG and IgA were enriched simultaneously, followed by a one-step denaturation, reduction, and alkylation. The obtained nanoLC-MS data were subjected to semiautomated, targeted feature integration and quality control. The combined and simplified protocol displayed high overall method repeatability, as assessed using pooled plasma and serum standards. Taking all samples together, 143 individual N- and O-glycopeptides were reliably quantified. These glycopeptides were attributable to 11 different peptide backbones, derived from IgG1, IgG2/3, IgG4, IgA1, IgA2, and the joining chain from dimeric IgA. Using this method, novel associations were found between IgA N- and O-glycosylation and age. Furthermore, previously reported associations of IgG Fc glycosylation with age in healthy individuals were confirmed. In conclusion, the new method paves the way for high-throughput multiprotein plasma glycoproteomics

Simultaneous Immunoglobulin A and G Glycopeptide Profiling for High-Throughput Applications

Immunoglobulin (Ig) glycosylation is recognized for its influence on Ig turnover and effector functions. However, the large-scale profiling of Ig glycosylation in a biomedical setting is challenged by the existence of different Ig isotypes and subclasses, their varying serum concentrations, and the presence of multiple glycosylation sites per Ig. Here, a high-throughput nanoliquid chromatography (LC)- mass spectrometry (MS)-based method for simultaneous analysis of IgG and IgA glycopeptides was developed and applied on a serum sample set from 185 healthy donors. Sample preparation from minute amounts of serum was performed in 96-well plate format. Prior to trypsin digestion, IgG and IgA were enriched simultaneously, followed by a one-step denaturation, reduction, and alkylation. The obtained nanoLC-MS data were subjected to semiautomated, targeted feature integration and quality control. The combined and simplified protocol displayed high overall method repeatability, as assessed using pooled plasma and serum standards. Taking all samples together, 143 individual N- and O-glycopeptides were reliably quantified. These glycopeptides were attributable to 11 different peptide backbones, derived from IgG1, IgG2/3, IgG4, IgA1, IgA2, and the joining chain from dimeric IgA. Using this method, novel associations were found between IgA N- and O-glycosylation and age. Furthermore, previously reported associations of IgG Fc glycosylation with age in healthy individuals were confirmed. In conclusion, the new method paves the way for high-throughput multiprotein plasma glycoproteomics