Major human milk oligosaccharides are absorbed into the systemic circulation after oral administration in rats.

Human milk oligosaccharides (HMO) are involved in many biological functions influencing infant health. Although HMO act locally at the intestine, recent evidence has demonstrated that HMO are partially incorporated into the systemic circulation of breast-fed infants. In the last few years, a large amount of research has been conducted using preclinical models to uncover new biological functions of HMO. The aim of this study was to evaluate the absorption and urine excretion of HMO in rats. We administered a single oral dose of the following HMO: 2'-fucosyllactose (2'-FL), 6'-sialyllactose and lacto-N-neotetraose at different concentrations to adult rats. The time course of absorption of HMO into the bloodstream and their appearance in urine was studied. Our results showed that rats, similar to human infants, are able to effectively absorb a portion of HMO from the intestine into plasma and to excrete them in urine. On the basis of this, we also conducted a specific kinetic absorption study with 2'-FL, the most predominant HMO in human milk, in 9-11-d-old rat pups. Our results confirmed that a significant amount of 2'-FL was absorbed into the systemic circulation and subsequently excreted in urine during lactation in rats in a dose-depended manner. We also found basal levels of these HMO in plasma and urine of adult rats as well as rat pups as a natural result of nursing. Our data suggest that the rat may be a useful preclinical model that provides new insights into the metabolism and functions of HMO.

Direct evidence for the presence of human milk oligosaccharides in the circulation of breastfed infants.

BACKGROUND: It has been hypothesized that human milk oligosaccharides (HMOs) confer systemic health benefits to breastfed infants; however, plausible mechanisms for some effects, such as systemic immunomodulation, require HMOs to access the bloodstream of the developing infant. While small concentrations of HMOs have been detected in the urine of breastfed infants there are no published studies of these oligosaccharides accessing the plasma compartment of breastfed infants. Here we determined the relative fractions of several ingested HMOs in infant urine and plasma. Plasma from formula-fed infants was used as a control. METHODS: Using gas chromatography/mass spectrometry (GC/MS), liquid chromatography/mass spectrometry/tandem mass spectrometry (LC/MS/MS), and high performance liquid chromatography (HPLC), we analyzed the urine and plasma from 17 healthy formula-fed infants and 16 healthy breast-fed infants (and the milk from their mothers). RESULTS: Multiple HMOs were detected in the urine and plasma of breastfed infants, but not in formula-fed infants. Levels of 2'-fucosyllactose (2'FL), 3FL and lacto-N-neotetraose (LNnT) in both plasma (r = 0.98, p<0.001; r = 0.75, p = 0.002; r = 0.71, p = 0.004) and urine (r = 0.81, p<0.001; r = 0.56, p = 0.026; NS) correlated significantly with concentrations in the corresponding breast milk. The relative fractions of HMOs were low, 0.1% of milk levels for plasma and 4% of milk levels for urine. Within the breastfed cohort, there were significant differences between secretor and nonsecretor groups in levels of several fucosylated HMOs. CONCLUSION: At least some ingested HMOs are absorbed intact into the circulation and excreted in the urine and their concentrations in these fluids correlate with levels of the corresponding mother's milk. While relative fractions of absorbed HMOs were low, these levels have been shown to have biological effects in vitro, and could explain some of the postulated benefits of human milk.

Absorption and urinary excretion of quercetin, rutin, and alphaG-rutin, a water soluble flavonoid, in rats.

Quercetin, rutin, alphaG-rutin (a water soluble flavonoid), and a mixture of rutin and alphaG-rutin were administered to rats by a single gastric intubation, and their absorption and urinary excretion were examined. The plasma and 24 h urinary levels of aglycons (quercetin and tamarixetin/isorhamnetin) were measured by HPLC after deconjugation with beta-glucuronidase/sulfatase treatment. alphaG-rutin was absorbed more rapidly than quercetin or rutin, and the plasma concentrations of quercetin and tamarixetin/isorhamnetin reached the highest peak level 30 min after dosing. Quercetin, rutin, and the mixture of rutin and alphaG-rutin showed the first peak level 8 h, 8 h, and 30 min after dosing, respectively. The area under the concentration-time curve (AUC) for quercetin in rats administered alphaG-rutin was approximately 4.5- and 2-fold higher than those in rats administered quercetin and rutin, respectively, and was almost the same as that in rats administered a mixture of rutin and alphaG-rutin. The highest 24 h urinary excretion was observed in alphaG-rutin-administered rats. These results suggest that alphaG-rutin is absorbed more efficiently than either quercetin or rutin and that a high plasma concentration can be maintained by supplying rutin and alphaG-rutin in combination.

Analysis of the antidiabetic drug acarbose by capillary electrophoresis.

This study describes the derivatization of the pseudooligosaccharide acarbose and its main metabolite, component 2, with 7-aminonaphthalene-1,3-disulfonic acid (ANDS) in human urine. Their efficient separation was possible by means of capillary zone electrophoresis, using a capillary tube of fused-silica containing 100 mM triethylammonium phosphate buffer, pH 1.5. On column laser-induced fluorescence allowed the detection of the pseudooligosaccharides in human urine in the nanomolar range. With this method, acarbose and component 2 were quantified in human urine after application of 300 mg of acarbose.

Identification of trisaccharide Xyl alpha 1-->3Xyl alpha 1-->3Glc in human urine.

Oligosaccharides in human urine were converted to pyridylamino (PA)-derivatives. From the PA-oligosaccharides, a saccharide that was chromatographically identical with a synthetic standard, Xyl-alpha 1-->3Xyl alpha 1-->3Glc-PA, was isolated by gel filtration and HPLC. Structure analysis showed that the saccharide was Xyl alpha 1-->3Xyl-alpha 1-->3Glc-PA. It is likely that Xyl alpha 1-->3Xyl alpha 1-->3Glc originated from such glycoconjugates as blood coagulation factors VII and IX, and protein Z.

Sialyl-alpha 2-6-mannosyl-beta 1-4-N-acetylglucosamine, a novel compound occurring in urine of patients with beta-mannosidosis.

Human beta-mannosidosis urine was fractionated by gel permeation chromatography on Bio-Gel P-2 and by high performance liquid chromatography on Partisil 10 SAX. Besides the disaccharide Man beta 1-4GlcNAc as the major component, a sialic acid-containing compound was detected in an amount of 10% compared to that of Man beta 1-4GlcNAc. Structural characterization of the oligosaccharide and of its reduced analogue by sugar composition analysis, methylation analysis, gas-liquid chromatography-mass spectrometry, and 500-MHz 1H NMR spectroscopy gave conclusive evidence for a novel urinary constituent: NeuAc alpha 2-6Man beta 1-4GlcNAc. This linear trisaccharide can be considered as the result of an alpha 2-6-sialylation of the major accumulating compound, Man beta 1-4GlcNAc. The hitherto unknown linkage between sialic acid and mannose was shown to be susceptible to sialidase digestion.

Source of the urinary maltose and maltotriose excreted during intravenous infusion of oligosaccharide solutions in young pigs.

Pigs infused with preparations of glucose oligosaccharides excrete sizeable quantities of maltose in urine despite the absence of maltose in the infused solution. Maltose infused without other oligosaccharides is well utilized. Our studies examined possible sources of excreted maltose. We first examined whether simultaneous infusion of larger oligosaccharides with maltose inhibits maltose utilization. Four young pigs were infused for four days with 20 g/d of a maltose-free oligosaccharide preparation to which tracer quantities of U-14C-maltose were added. Urinary excretion of maltose-plus-maltotriose increased significantly (P less than .05) from a mean +/- SD baseline value of .01 +/- .01 g/d to an overall four-day mean value of 1.33 +/- 0.47 g/d. However, only 10.7 +/- 0.78% of infused 14C-maltose was excreted, indicating that simultaneous infusion of larger oligosaccharides did not inhibit maltose utilization. The second study examined whether maltotriose present in the oligosaccharide mixture was the source of excreted maltose. Four young pigs were infused for three days with 20 g/d of a special preparation of oligosaccharides containing only trace quantities of maltose and maltotriose. Urinary maltose plus maltotriose excretion increased significantly (P less than .05) from a mean +/- SD baseline value of .01 +/- .01 g/d to 1.65 +/- 0.41 g/d during oligosaccharide infusion. The data suggest that excreted maltose and maltotriose arise from the catabolism of larger oligosaccharides.

Determination of the structure of a fucose-containing trisaccharide monophosphate isolated from human pregnancy urine.

A new acidic oligosaccharide, isolated from the urine of a pregnant woman by gel filtration and ion-exchange chromatography, was shown on the basis of sugar analysis, methylation analysis, exo-glycosidase digestion, e.i.-m.s., f.a.b.-m.s., and n.m.r. spectroscopy to have the following structure: (Formula: see text).

Maltotriose and maltotetraose excreted in urine following intravenous administration of maltose to human volunteers.

To determine the extent of maltose excreted into the urine, sugar substances present in the urine following intravenous infusion of maltose were analyzed. Maltose, glucose, maltotriose and maltotetraose in the urine were detected by gas chromatography and identified by mass spectrometric analysis. The total amounts of sugar substances excreted after 10 per cent maltose solution given at three different infusion rates were calculated. The excreted amounts of maltotriose and maltotetraose increased in a dose and time dependent manner. As these compounds were not detected in the plasma either during or after the administration of maltose, the kidney probably plays a role in the biosynthesis of maltotriose and maltotetraose. Studies on the organ homogenates of the rabbit showed that the enzyme activity for the biosynthesis of maltotriose from maltose was mainly in the kidney. The glucose excreted into the urine probably originates from maltose catalyzed to glucose, mainly by the action of kidney maltase. As the rate of excretion of sugar substances increased in a dose dependent manner, adequate infusion rates of maltose should be less than 0.5 g/kg/hour.

Urinary levels of blood group A trisaccharide: observations in two siblings with neuronal ceroid lipofuscinosis.

A readily detectable carbohydrate in the urine of two siblings with neuronal ceroid lipofuscinosis was found to be the blood group A trisaccharide. One child expired before blood typing was done while the other sibling was AB. This latter child excreted greater amounts of the trisaccharide than a group of blood type A subjects with different diagnoses when all the subjects were receiving whole or skim milk by nasogastric feedings. While the relevance of this observation to neuronal ceroid lipofuscinosis is unknown, it has been shown that the trisaccharide may be a major urinary carbohydrate depending on diet and blood type.

Determination of carbohydrates in urine by high-performance liquid chromatography and optical activity detection.

Improvements in a detector for liquid chromatography based on optical activity of the components have led to a detectability of 100 ng. This allows the simultaneous determination of six naturally occurring carbohydrates in 100-microliters samples of human urine, which is injected directly except for a simple deionization step. The reproducibility and reliability of this method should allow better insight into the relation between urinary sugars and physiological conditions.

Increased excretion of two sialic acid-containing trisaccharides in the urine of patients with rheumatoid arthritis.

The urinary excretion of sialic acid-containing trisaccharides in patients with active rheumatoid arthritis was studied. Sialyl-lactose and sialyl-N-acetyllactosamine were identified and their excretion patterns studied by thin layer and gas chromatography. The urinary output of sialyl-lactose was greater in patients with active rheumatoid arthritis (48.2 +/- 6.1 mg/24 h, SEM, n = 6) than in healthy subjects (19.8 +/- 3.7 mg/24 h, SEM, n = 5; P less than 0.01). The excretion of sialyl-N-acetyllactosamine was also higher in the rheumatoid group (18.5 +/- 2.1 mg/24 h, SEM, n = 6) than in the controls (11.1 +/- 1.2 mg/24 h, SEM, n = 5; P less than 0.05). The qualitative excretion patterns of the sialyl-oligosaccharide fraction were similar for the two groups as judged from the thin layer chromatograms. Correlating the results with the clinical state of the patients with rheumatoid arthritis suggests that the urinary level of the sialyl-oligosaccharides reflects the activity of the disease. A proposed mechanism for the increased excretion of sialic acid-containing trisaccharides in rheumatoid arthritis is presented.

Excretion of neuraminic acid-containing trisaccharides in the urine during pregnancy.

The excretion of neuraminic acid-containing trisaccharides in the urine during the course of normal pregnancy was studied. Neuraminyl-lactose and neuraminyl-galactosyl-(l led to 4)-N-acetylglucosamine were identified, and their excretion patterns were analyzed by thin-layer and gas chromatography. A progressive increase in the outputs of these acidic oligosaccharides was observed during the course of pregnancy in all cases studied. Neuraminyl-lactose excretion increased threefold, from 13.7 +/- 1.75 to 37.1 +/- 2.56 mg/24 h, and neuraminyl-galactosyl-N-acetylglucosamine twofold, from 8.4 +/- 1.27 to 15.3 +/- 2.21 mg/24 h (Mean +/- S.E.).