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Glycobiology Advance Access originally published online on February 22, 2006
Glycobiology 2006 16(6):477-487; doi:10.1093/glycob/cwj092
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© The Author 2006. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Incorporation of orally applied 13C-galactose into milk lactose and oligosaccharides

Silvia Rudloff1,2, Susanne Obermeier3, Christian Borsch4, Gottfried Pohlentz5, Rudolf Hartmann6, Herbert Brösicke7, Michael J. Lentze8, and Clemens Kunz3,4

2 Department of Pediatrics, University of Giessen, Feulgenstr. 12, 35392 Giessen, Germany; 3 Research Institute of Child Nutrition Dortmund, 44225 Dortmund, Germany; 4 Institute of Nutritional Sciences, University of Giessen, 35392 Giessen, Germany; 5 Institute of Medical Physics and Biophysics, University of Münster, 48149 Münster, Germany; 6 Institute of Physiological Chemistry, University of Bonn, 53113 Bonn, Germany; 7 Virchow-Clinics, 10117 Berlin, Germany; and 8 Center of Pediatrics, University of Bonn, 53113 Bonn, Germany

1 To whom correspondence should be addressed; e-mail: silvia.rudloff{at}ernaehrung.uni-giessen.de

Received on August 5, 2005; revised on February 14, 2006; accepted on February 15, 2006


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Materials and methods
 Conflict of interest statement
 Acknowledgments
 References
 
Biosynthesis and functions of human milk oligosaccharides (HMO) are not well known. A typical housekeeping enzyme, ß1,4-galactosyltransferase, links galactose to glucose to form lactose which is then used as backbone for the assembly of HMO. We investigated whether milk lactose and HMO may be labeled in vivo by an orally given 13C-galactose bolus. Eleven exclusively breastfeeding mothers were given a 13C-galactose bolus at the end of their breakfast. Milk and urine samples from each nursing up to 36 h were analyzed for carbohydrate composition by high-performance thin-layer chromatography, high-pH anion-exchange chromatography, and fast atom bombardment mass spectrometry. 13C enrichment of milk fractions, urinary carbohydrates, lactose, and oligosaccharides as well as of breath CO2 was determined by isotope ratio mass spectrometry. Up to 10% of the orally given galactose bolus was directly transported to the mammary gland and incorporated into milk components. Characteristic for most milk samples was the appearance of two 13C-peaks, the first immediately after the 13C-bolus was taken and the second on the next morning. The highest 13C enrichment was found in lactose followed by neutral and acidic oligosaccharides. In breath samples, the 13C-excretion followed the same pattern as in milk. 13C nuclear magnetic resonance of isolated lactose revealed 13C only at C1-atom of galactose and C1-atom of glucose. This label was without any exception at the same position as the 13C-label of the orally applied galactose. Neutral and acidic HMO can easily be 13C-labeled in vivo which facilitates investigations of their metabolic fate in infants.

Key words: 13C-lactose / 13C-oligosaccharides / fast atom bombardment mass spectrometry / human milk / isotope ratio mass spectrometry


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Materials and methods
 Conflict of interest statement
 Acknowledgments
 References
 
The human mammary gland is a unique tissue, because it is thought to be the only organ where lactose and lactose-derived oligosaccharides can be synthesized (Brew and Hill, 1975Go; Kobata et al., 1978Go; Charron et al., 1998Go). A ß1,4-galactosyltransferase as housekeeping enzyme links galactose to glucose instead of GlcNAc to form lactose which is then used for the assembly of human milk oligosaccharides (HMO) present in concentrations of about 5–10 g/L (Kunz et al., 2000Go). The quantity and the pattern of HMO are affected by the expression of specific glycosyltransferases in the mammary gland. Genes encoding for the Lewis type blood group and the secretor status determine the presence of {alpha}1-2-, {alpha}1-3- and/or {alpha}1-4-fucosylated core structures of HMO (Watkins and Morgan, 1957Go; Egge, 1992Go). In addition, different patterns of sialylation, that is, {alpha}2-3- and/or {alpha}2-6-linkages of N-acetylneuraminic acid (NeuAc), increase the variability of HMO to a number of >150 compounds characterized so far (Egge et al., 1983Go; Stahl et al., 1994Go; Kunz et al., 2000Go).

HMO are discussed to play an important role in the infants’ defense against certain infections and during inflammatory processes (Zopf and Roth, 1996Go; Sharon and Ofek, 2000Go). For example, they may promote the growth of a non-pathogenic intestinal microflora or function as receptor analogs preventing the epithelial attachment of pathogenic micro-organisms. Recently, it has been shown that they inhibit leukocyte adhesion to activated endothelial cells, and they may influence neutrophil platelet interactions (Bode, Kunz et al., 2004Go; Bode, Rudloff et al., 2004Go).

Despite an increasing number of animal and preclinical studies (Idäänpään-Heikkil et al., 1997Go; Mysore et al., 1999Go), the in vivo evidence for specific functions of HMO in the infant is still lacking. To perform functional studies in humans, it would be of great advantage to achieve an in vivo labeling of milk oligosaccharides to be able to follow their metabolic fate in the recipient infant.

Thus, we used a straightforward approach and orally applied 13C galactose to lactating mothers investigating whether or not this monosaccharide is directly used for the biosynthesis of the large amounts of lactose (50–70 g/L) as well as of oligosaccharides (5–10 g/L), without being metabolized by the liver first. Because (a) milk biosynthesis can be turned on to a maximum within a few minutes, (b) the amount of milk produced by a women can reach up to several liters a day, and (c) galactose but not glucose is a main component in HMO, we assumed that it could be an advantage to offer galactose instead of glucose to the lactating mammary cell. Previously we explained our hypothesis that orally given galactose is incorporated into milk lactose and oligosaccharides (Kunz et al., 2000Go). We exemplified the potential of applying stable isotopes to lactating mothers and monitoring the metabolic fate in their infants by reporting the data from one mother–infant pair. Also, we demonstrated earlier analyzing milk from one woman that the application of 2 g 13C-galactose not only leads to a 13C-enrichment of whole milk but of lactose as well as of neutral and acidic oligosaccharide fractions (Obermeier et al., 1999Go). As an extension of our previous work, we describe here for the first time the applied methods, we increased the amount of the 13C-bolus to 4 g 13C-galactose, and we demonstrate the efficacy of the method in 11 other mothers. These women received a 13C-galactose bolus with either 2 or 4 g 13C galactose immediately after breakfast. In addition, we wanted to quantitate the amount of galactose which is directly incorporated into lactose and lactose-derived oligosaccharides. Moreover, if the 13C enrichment of oligosaccharides was high enough, investigations on the metabolic fate of a particular component in the recipient infants and on the amount an infant receives per day should be possible in subsequent studies.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Materials and methods
 Conflict of interest statement
 Acknowledgments
 References
 
13C enrichment of whole milk and 13CO2-breath tests
After the oral 13C-galactose bolus had been given, there was an immediate, marked 13C enrichment of the whole milk in the first 4–8 h (Figure 1). Then, a fast decline of the {delta}13CPDB values followed. Individual differences were observed in the intensities of the first 13C peaks and the presence of a second 13C peak in the morning of the next day. In milk from four women, the 13C enrichment on the second day was as high or even more pronounced than that on the first day (Figure 1).


Figure 1
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  		Fig. 1. 13C-enrichment of whole milk from 11 women during the first 36 h after the oral intake of a galactose bolus consisting of either 2 g 13C-galactose + 25 g galactose (subjects with filled lines) or 4 g 13C-galactose + 23 g galactose (subjects with interrupted lines). The {delta}13CPDB({per thousand})-values of each sample are corrected over the baseline values of each respective sample obtained at time point 0, immediately before the galactose intake. The bolus was taken after breakfast which varied between 8:00 and 9:30 AM.

 

13C enrichment of breath was determined for nine of 11 mothers by analyzing 13CO2. The 13CO2 exhalation over 36 h resulted in a similar curve compared to the 13C enrichment of whole milk (Figure 2).


Figure 2
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  		Fig. 2. 13C-CO2 exhalation over a period of 36 h after the intake of the 13C-galactose bolus. The {delta}13CPDB({per thousand})-values of each sample are corrected over the baseline values (see also legend to Figure 1).

 

Cumulative 13C enrichment of whole milk, breath, and urine
The 13Ccum enrichment in milk on day 1 was between 3.3 and 10.5% of the oral 13C dose (Table I). Table I also shows the 13Ccum enrichment in breath and urine of the mothers on the first day in a range between 14.8 and 40.9% and 0.7 and 1.4%, respectively.


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  		Table I. Cumulative 13C enrichment of breath, milk, and urine (% of oral bolus)

 

13C enrichment of milk fractions
Due to the high {delta}13CPDB in whole milk, we addressed the question whether or not a specific 13C enrichment occurs in one particular milk fraction. As a representative example, Figure 3 shows that {delta}13CPDB in the carbohydrate fraction from the first milk samples of one mother was more than twice as high as in whole milk. The course of the 13C enrichment of the carbohydrate fraction was in parallel to that of whole milk. The 13C enrichment of the protein fraction, however, was less than half that of whole milk. In the fat fraction, the {delta}-values were found to be almost baseline level.


Figure 3
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  		Fig. 3. 13C enrichment of whole milk and of milk fractions from one woman after an oral galactose bolus (2 g 13C-galactose and 25 g galactose) was given (see also legend to Figure 1). Milk aliquots were collected from each suckling over a period of 36 h. Whole milk {blacksquare}, fraction with carbohydrates {blacktriangleup}, proteins {square}, and fat {circ}.

 

Composition and 13C enrichment of the carbohydrate fraction
To characterize milk carbohydrates and to determine their 13C enrichment, we defatted, deproteinized, and separated whole milk by Sephadex G25 gel filtration into five fractions with components of various molecular weights (Figure 4). Investigations of the composition of these fractions by high-performance thin-layer chromatography (HPTLC) (Figure 4, inset), high-pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) (Figure 5), and fast atom bombardment mass spectrometry (FAB-MS) revealed the following composition of the Sephadex G25 fractions: mainly acidic oligosaccharides in fractions 1 and 2, mainly complex neutral components in fraction 3, neutral oligosaccharides in fraction 4, and mainly lactose in fraction 5.


Figure 4
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  		Fig. 4. Sephadex G25 gel filtration of a defatted and deproteinized milk sample and thin layer chromatography (inset) of the resulting carbohydrate fractions. Carbohydrates were visualized by spraying the HPTLC plates with an orcinol reagent according to Kunz et al., 1996Go.

 

Figure 5
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  		Fig. 5. HPAEC-PAD of Sephadex G25 fractions of a milk sample and their 13C enrichment. The retention times for the neutral components (fraction 3–5) are 3–17 min and for the acid components (fractions 1 and 2) above 17 min. The fractions consist of the following main components 1 Lac, 2 Fuc2-LNT, 3 Fuc{alpha}1–3/2Lac, 4 LNT, 5 LNFP I, 6 multiple fucosylated tetraoses, hexaoses etc, 7 monofucosylated hexaoses, octaoses, and decaoses, 8 NeuAc-LNT (LSTc), 9 NeuAc{alpha}2-6Lac, 10 NeuAc{alpha}2-3Lac, 11 NeuAc-LNT (LSTa/b), 12 NeuAc2-LNT and multiple sialylated components.

 
{delta}13CPDB of each Sephadex G25 fraction of all milk samples during 36 h from all participants revealed in most samples the highest {delta}13C enrichment in lactose followed by fractions with mainly neutral oligosaccharides and acidic components (Figure 6). However, in some samples the 13C enrichment of neutral and acidic components was similar to or even higher than that of lactose (Figure 6).


Figure 6
Figure 6
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  		Fig. 6. 13C enrichment of acidic oligosaccharides (fractions 1 and 2), neutral oligosaccharides (fraction 3 and 4) and lactose (fraction 5) after an oral galactose bolus (2 g 13C-galactose + 25 g galactose or 4 g 13C-galactose + 23 g galactose) was given. Milk samples were collected over a period of up to 36 h (see also legend to Figure 1).

 

Isotope ratio mass spectrometry of 13C-lactose and 13C-oligosaccharides
Due to the high 13C content not only in lactose but also in neutral and acidic oligosaccharides, we collected single peaks after HPAEC-PAD and determined their structure by FAB-MS as well as their {delta}13CPDB by isotope ratio mass spectrometry (IRMS). Especially those fractions which contain lacto-N-tetraose and fucosyl-lacto-N-tetraose were highly 13C-enriched compared with the baseline values (–26{per thousand}). Even more complex components such as difucosyl-lacto-N-tetraose, fucosyl-lacto-N-hexaose or difucosyl-lacto-N-hexaose were highly 13C-enriched.

Incorporation of 13C into lactose and quantitation of the contribution of orally ingested galactose to lactose biosynthesis
To investigate how much of the oral 13C-galactose is directly incorporated into milk lactose, we performed 13C-NMR studies. To find out on which position within the lactose molecules the 13C-label was located, we isolated 10–20 mg lactose from each milk sample of all 11 women throughout 36 h. In all samples, the 13C atom was present only in the C1-position of galactose and C1-position of glucose within the lactose molecule. An example is given in Figure 7 and Table II. The amount of oral galactose which was directly incorporated into lactose was calculated for the first milk sample, after the 13C-galactose bolus was given. The individual variation in the 11 mothers was between 0.4 and 19.4%.


Figure 7
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  		Fig. 7. 13C-NMR analysis of a lactose sample isolated from the first milk sample collected after the 13C-galactose bolus (in black) was given and of a lactose standard (in grey). As reference the C-4-signal of glucose was set to 1.00. Because glucose is localized at the reducing end of the lactose molecule, in the 13C-NMR experiment it appeared twice, as {alpha}- and as ß-Glc (see corresponding Table II).

 

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  		Table II. 13C-NMR analysis of an isolated lactose sample. 13C chemical shifts are given in ppm (see also corresponding Figure 6)

 

Urine analysis
The cumulative 13C-elimination rate in urine was calculated to be 0.8–1.0% of the ingested dose. Because the excretion of enzymatically determined galactose content in urine was in parallel to the corresponding 13CPDB values, 13C most likely originates from the ingested free 13C-galactose.


   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Materials and methods
 Conflict of interest statement
 Acknowledgments
 References
 
Human milk oligosaccharides are considered to have anti-inflammatory and anti-infective capacities (Zopf and Roth, 1996Go; Sharon and Ofek, 2000Go; Kunz et al., 2000; Bode, Kunz et al., 2004Go; Bode, Rudloff et al., 2004Go). However, there are only few data available addressing metabolic questions in lactating mothers and their infants. An in vivo labeling of milk oligosaccharides would enable such studies. Therefore, we first applied 1 or 2 g 13C-labeled galactose together with 25 g non-labeled galactose to lactating mothers (Obermeier et al., 1999Go and unpublished data). In the course of the study, we increased the ratio of 13C-galactose/galactose (4 g 13C-galactose + 23 g galactose) with the intention to increase the 13C enrichment of milk which would be an advantage in succeeding metabolic studies.

The application of 25 g non-labeled galactose is based on earlier observations by Lundblad and coworker that the intake of 25 g galactose or 50 g of lactose but not of 25 g glucose by healthy men leads to the urinary excretion of blood group active pentasaccharides some of which contain lactose as backbone (Lundblad et al., 1973Go). This phenomenon can still not be explained, because the lactating mammary gland is considered to be the only organ capable of lactose synthesis; however, since that time, it has also been intriguing to speculate that orally given monosaccharides other than glucose may directly affect glycoconjugate biosynthesis. Recent studies support these observations. In patients with congenital disorders of glycosylation (CDG) syndrome Ib, an oral mannose treatment leads to a significant improvement of the clinical conditions (Niehues et al., 1998Go). A further example is the oral application of fucose to a patient with CDG-IIc revealing improved fucosylation of glycoproteins and reduced recurrent infections (Etzioni et al., 2000Go).

Applying 13C-galactose to lactating mothers, we found that in milk of nine of 11 women, two 13C-peaks were observed; the first one shortly after the galactose bolus was taken in and the second one in the morning of the next day. We suggest that the rise in 13C on day 2 is due to the mobilization of 13C-glucose from liver glycogen. This assumption is also based on studies regarding liver glycogen metabolism (Shulman et al., 1990Go).

Comparing the 13C enrichment of whole milk and 13CO2 in breath, most of the ingested galactose is obviously used in metabolic processes as can be concluded from the increased exhalation of 13CO2 (Figure 2). Up to 40% of the oral 13C dose can be detected in breath (Table I). The large individual variation may be due to a different turnover, nutritional status, or metabolic activity of the liver. The cumulative 13C enrichment in milk varies between 3.3 and 10.5% calculated for the first 15–20 h. After separating milk into different fractions, we found the highest enrichment in carbohydrates. With regard to the 13C enrichment of carbohydrate fractions, we observed that not only lactose but also neutral and acidic oligosaccharides are highly 13C-enriched. In some samples the enrichment was similar or even higher than that of lactose (Table II). Considering the complex structures of oligosaccharides, there might be specific mechanisms ensuring a preferential incorporation of 13C-galactose into oligosaccharides. This mechanism obviously guarantees the conversion of glucose into galactose even 36 h after the 13C-bolus was given.

Due to the large amount of lactose in milk, we were able to isolate 10–20 mg from each sample to perform 13C-NMR measurements. The analysis of more than 200 samples convincingly demonstrated without any exception that only the C1-atom of galactose and C1-atom of glucose within the lactose moiety represented 13C. Because the 13C-galactose ingested by the women was 99% labeled with 13C at the C1-position, the 13C-labeled galactose must have been used directly or after epimerization of the 13C-galactose to 13C-glucose for the biosynthesis of milk carbohydrates. A complete epimerization of the 13C-galactose into 13C-glucose before the incorporation seems unlikely for the following reasons: in seven of the 11 women we observed a greater enrichment of C1-galactose in comparison with C1-glucose by 13C-NMR in the first milk sample. In addition, in an experiment prior to the current study, 13C-NMR of lactose from milk of a subject with only 1 g 13C-galactose revealed a weak 13C enrichment within the galactose moiety only, whereas glucose remained unlabeled. Also, preliminary 13C-NMR of isolated lacto-N-tetraose revealed only a 13C-atom on position 1 of both galactose moieties but not on glucose or GlcNAc (data not shown).

Taken together, these results expand our current information about oligosaccharide biosynthesis. Besides glucose which obviously plays a unique cellular role in the synthesis of N-linked glycans, (Spiro, 2000Go) galactose seems to be important as well in special situations such as lactation. Certainly, glucose itself is also used for the biosynthesis of carbohydrates as has been found in recent studies investigating lactose biosynthesis (Sunehag et al., 2002Go). However, the question which arises from our study is whether or not it is of greater advantage from an energetic and physiological point of view to reserve glucose as a substrate for the maintenance of glucose homeostasis and intermediate metabolism. Our data are in line with the conclusion of Sunehag et al. (2002)Go studying lactose production that although adequate carbohydrate supply is important for lactogenesis, processes independent of glucose availability are required to maintain lactose production.

In conclusion, this study provides evidence for the preferred incorporation of orally given galactose into milk lactose and oligosaccharides in lactating women. A surprisingly high 13C-enrichment not only of lactose but also of neutral as well as of acidic oligosaccharides can be achieved by an in vivo labeling through the oral ingestion of 13C-galactose. Thus, a physiological approach for labeling milk components facilitates further studies on their metabolic fate in the recipient infants.


   Materials and methods
Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
Conflict of interest statement
 Acknowledgments
 References
 
Subjects and study design
Exclusively breast feeding women (n = 11; 1–5 months postpartum) participated in the study. Some dietary restrictions, that is, the avoidance of naturally 13C-rich food (e.g. corn, pineapple, fish) were given. Age and body weight of the mothers and their term infants as well as the number of infants were recorded. The mothers were asked not to drink milk for breakfast on the study day and to keep a brief record of their food intake during the study period. All women volunteered to participate in the studies which were approved by the ethical committees of the University in Dortmund and the University of Giessen.

Between 8:00 and 9:30 AM immediately after breakfast, the women orally ingested a galactose bolus consisting of 25 g galactose + 2 g 13C-galactose (n = 7) or 23 g galactose + 4 g 13C-galactose (n = 4). The purity of 13C-galactose (D-galactose; 1 – 13C, 99%) from Promochem (Wesel, Germany) or from Eurisotop (Saint-Aubin Cedex, France) was determined by NMR to be more than 99%. The galactose bolus was ingested after dissolving in a total of 40–50 mL tap water.

Milk, urine, and breath sampling of the women
A milk sample (5–10 mL) was collected immediately before the galactose bolus was given (baseline value) and at the beginning of each nursing during the following 36 h. At the same time, a breath sample was also taken by the mother, and her urine was collected in 3–4 h intervals.

Separation of milk fractions and characterization of milk carbohydrates
The separation of whole milk into fractions with fat, proteins, and carbohydrates followed by the isolation of lactose from oligosaccharides has been achieved by ultracentrifugation, acetone treatment and gel filtration chromatography as described earlier (Kunz and Lönnerdal, 1989Go; Kunz et al., 1996Go). Then, neutral and acidic HMO were characterized by chromatographic and mass spectrometry as follows.

High-performance thin-layer chromatography
An aliquot of each Sephadex G25 fraction (4 µL) was subjected to silica-HPTLC plates together with mono- and oligosaccharide standards. They were developed twice in butanol/acetic acid/H2O (2.5/1/1; v/v/v) or butanol/ethanol/H2O/acetic acid/pyridine (5/50/15/1.5/5; v/v/v/v/v) and then sprayed with orcinol for carbohydrate detection (0.1% orcinol in 20% H2SO4) or ninhydrin reagent (0.3 g ninhydrin in 95 mL 2-propanol containing 5 mL 96% acetic acid) (Kunz et al., 1996Go).

High-pH anion-exchange chromatography with pulsed amperometric detection
The system used for HPAEC-PAD of neutral and sialylated oligosaccharides consisted of a Dionex Bio-LC gradient pump, Carbo Pac PA-1 column (250 x 4.6 mm ID) equipped with a guard column and a Model PAD 2 detector (Dionex, Sunnyvale, CA) (Kunz et al., 1996Go). Neutral and acidic oligosaccharides were analyzed by HPAEC-PAD using the following conditions: Eluent A, 100 mM NaOH; Eluent B, 100 mM NaOH and 250 mM Na-acetate. The elution program began with 3 mL of buffer A, followed by a gradient of up to 100 % buffer B in 30 min. A re-equilibration volume of 5 mL buffer A was chosen. The flow-rate of 1.0 mL/min was used, and 25 µL of 1 or 4 mg/mL solutions were injected. Molar response factors was determined by injecting 3–6 times equimolar amounts of each oligosaccharide as described previously.

Fast atom bombardment mass spectrometry
Oligosaccharides (50–100 µg) were dissolved in 500 µL of pyridine/acetic anhydride (1/1; v/v). The mixture was stirred at room temperature overnight. Then the solvent was evaporated under nitrogen, and the residue was used for mass spectrometric analysis without further purification. The FAB-MS analysis was carried out on a VG-ZAB-T four sector instrument (Fisons Instruments, Manchester, UK); cesium was used for atom bombardment. The applied acceleration voltage was 8 kV. Thioglycerol/m-nitrobenzylalcohol (1/1; v/v) was used as matrix to which 1 µL of a homogeneous sample solution in chloroform/methanol (1/1; v/v) was added. For spectra recording, positive ions (FAB+) were detected after the first electrostatic analyzer on the second photomultiplier point detector. The spectra were run in a mass range from 100 to 2500 atom mass units with a scan rate of 8 s per decay and up to 10 scans were accumulated. For data acquisition and processing, the software Opus V3 was used (Pohlentz et al., 1994Go).

Determination of total organic carbon
Determination of total organic carbon (TOC) was carried out using a Total Carbon Analyzer (TOC-5000/5050; Shimadzu Europa GmbH, Duisburg, Germany). Briefly, 10 mL diluted milk (1:500) or urine (1:200) was combusted at 680°C. CO2 produced originating from total carbon (TC) was dried and then, after removal of the halogens by an absorbing material, measured with a non-disperse infrared gas analyzer (NDIR) at a wavelength of 4.3 µm. To determine the amount of inorganic carbon (IC), we added an acidic IC reagent (H3PO4) to the sample, and the CO2 released was detected by the NDIR. TOC (mg/L) was calculated by subtracting IC from TC. The absolute amount of TOC (g) in each sample was determined according to the equation TOC (g) = Vsa (L) x TOC (mg/L) x 10–3, with Vsa as sample volume.

For milk, the Vsa was the volume of each feeding derived by weighing the infant before and after nursing and dividing this weight by the specific density of milk ({rho} = 1.031 g/mL). For urine, Vsa was the excreted volume over the collection period.

Determination of 13C enrichment by isotope ratio mass spectrometry
13C enrichment was determined as {delta}13CPDB measured in duplicate in whole milk, milk carbohydrates, urine, and urinary carbohydrates by IRMS (Delta S, Finnigan MAT, Bremen, Germany) after total combustion at 1020°C (NA 1500 Series 2, Carlo Erba Instruments, Rodano, Italy). Breath samples were analyzed in a BreathMat IRMS (Finnigan MAT). 13C elimination rates were then quantified as described below.

Mass spectrometric 13C analysis by IRMS
The 13C content of a sample is expressed as delta value ({delta}13 CPDB) ({per thousand}) which is the relative difference between the 13C/12C ratio of a sample (sa) and the international Pee Dee Belemnite (PDB) standard with a (13C/12C)PDB ratio of 0.0112372 (Schoeller et al., 1980Go).



Formula

where 13Rsa = (13C/12C)sa is the isotope ratio of the unknown sample.

Data evaluation was performed with the Finnigan MAT ISODAT V5.2 software (Finnigan MAT) taking into account the 17O content of the sample and the blank correction for the carbon of the tin cups (Craig, 1957Go). For an easier comparison of the data, the results were expressed as {Delta}{delta}13CPDB (corrected for the 13C/12C ratio at baseline). The accepted standard deviation was <0.2{per thousand} for total urine and isolated carbohydrates and <0.7{per thousand} for total milk. The accepted deviation for breath sample analyses was <0.3{per thousand}.

Calculation of the cumulative 13C enrichment in milk and urine
To determine the 13C enrichment in milk and urine, TOC and the 13C content (atom%) for each sample were measured. Then, the excreted amounts of 13C were calculated, taking into account the baseline 13C values and the 13C bolus given.

Using this 13C-at%sa and the TOC of a sample, we then calculated the absolute amount of 13C of each sample (sa) as follows:

Formula

Calculation of the cumulative 13C exhalation
The percentage recovery of the ingested 13C amount in breath (13Ccum) was calculated using the equation shown above. Thereby, the exhaled 13C amount is defined as


Formula

with {Delta}{delta}13Ccum being the cumulative 13C exhalation calculated according to the trapezoidal rule described by Schoeller et al. (1980)Go, and CO2-P as the individual CO2 production of the subject. CO2-P was estimated taking into account the basal metabolic rate, age, weight, height, and activity levels of the subject according to Schofield (1985)Go.

13C nuclear magnetic resonance analysis
Twenty milligrams of lactose isolated from human milk or a lactose standard was dissolved in deuterated water (D2O). After the addition of 5 µL 2 M NH4OH, the samples were analyzed in a proton decoupled 13C experiment at 25°C with 1024 scans in a magnetic field of a strength of 11.74 Tesla (AMX 500 NMR-Spektrometer, Bruker, Karlsruhe, Germany). The processing of the 13C experimental results was performed using the software XWinNMR (Bruker). To determine 13C enrichment and to take into account the different relaxation times of single 13C atoms, the analysis was referred to a lactose standard. Here, the C-4 signals were arbitrarily set to 1.00. This means that the intensity of the C-4 signal corresponded to the natural 13C abundance of 1.1%.

Determination of galactose and lactose content
Galactose and lactose contents of milk and urine were photometrically determined using a colorimetric kit from Boehringer Mannheim (Mannheim, Germany).


   Conflict of interest statement
Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Conflict of interest statement
Acknowledgments
 References
 
None declared.


   Acknowledgments
Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Conflict of interest statement
 Acknowledgments
References
 
We appreciate very much the contribution of all mothers in participating in this study. Supported by the Deutsche Forschungsgemeinschaft (Ku 781/8–3).


   Abbreviations
 
13C-NMR, 13C-nuclear magnetic resonance spectroscopy; FAB-MS, fast atom bombardment mass spectrometry; Fuc2-LNT, difucosyl-lacto-N-tetraose; Fuc{alpha}1–3/2Lac, fucosyl-lactose; GlcNAC, N-acetylglucosamine; HMO, human milk oligosaccharides; HPAEC-PAD, high-pH anion exchange chromatography with pulsed amperometric detection; HPTLC, high-performance thin-layer chromatography; IC, inorganic carbon; IRMS, isotope ratio mass spectrometry; LNFP I, lacto-N-fucopentaose I; LNT, lacto-N-tetraose; NDIR, non-disperse infrared gas analyzer; NeuAc2-LNT, disialyl-lacto-N-tetraose; NeuAc-LNT, sialyl-lacto-N-tetraose; NeuAc{alpha}2-3Lac, sialyl-lactose; NeuAc{alpha}2-6Lac, sialyl-lactose; TC, total carbon; TOC, total organic carbon


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Conflict of interest statement
 Acknowledgments
 References
 
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