Ellen G. H. M. van den Heuvel

A randomised crossover study investigating the effects of galacto-oligosaccharides on the faecal microbiota in men and women over 50 years of age

Faecal microbial changes associated with ageing include reduced bifidobacteria numbers. These changes coincide with an increased risk of disease development. Prebiotics have been observed to increase bifidobacteria numbers within humans. The present study aimed to determine if prebiotic galacto-oligosaccharides (GOS) could benefit a population of men and women of 50 years and above, through modulation of faecal microbiota, fermentation characteristics and faecal water genotoxicity. A total of thirty-seven volunteers completed this randomised, double-blind, placebo-controlled crossover trial. The treatments - juice containing 4 g GOS and placebo - were consumed twice daily for 3 weeks, preceded by 3-week washout periods. To study the effect of GOS on different large bowel regions, three-stage continuous culture systems were conducted in parallel using faecal inocula from three volunteers. Faecal samples were microbially enumerated by quantitative PCR. In vivo, following GOS intervention, bifidobacteria were significantly more compared to post-placebo (P = 0·02). Accordingly, GOS supplementation had a bifidogenic effect in all in vitro system vessels. Furthermore, in vessel 1 (similar to the proximal colon), GOS fermentation led to more lactobacilli and increased butyrate. No changes in faecal water genotoxicity were observed. To conclude, GOS supplementation significantly increased bifidobacteria numbers in vivo and in vitro. Increased butyrate production and elevated bifidobacteria numbers may constitute beneficial modulation of the gut microbiota in a maturing population.

The role of menaquinones (vitamin K 2 ) in human health

Recent reports have attributed the potential health benefits of vitamin K beyond its function to activate hepatic coagulation factors. Moreover, several studies have suggested that menaquinones, also known as vitamin K2, may be more effective in activating extra-hepatic vitamin K-dependent proteins than phylloquinone, also known as vitamin K1. Nevertheless, present dietary reference values (DRV) for vitamin K are exclusively based on phylloquinone, and its function in coagulation. The present review describes the current knowledge on menaquinones based on the following criteria for setting DRV: optimal dietary intake; nutrient amount required to prevent deficiency, maintain optimal body stores and/or prevent chronic disease; factors influencing requirements such as absorption, metabolism, age and sex. Dietary intake of menaquinones accounts for up to 25% of total vitamin K intake and contributes to the biological functions of vitamin K. However, menaquinones are different from phylloquinone with respect to their chemical structure and pharmacokinetics, which affects bioavailability, metabolism and perhaps impact on health outcomes. There are significant gaps in the current knowledge on menaquinones based on the criteria for setting DRV. Therefore, we conclude that further investigations are needed to establish how differences among the vitamin K forms may influence tissue specificities and their role in human health. However, there is merit for considering both menaquinones and phylloquinone when developing future recommendations for vitamin K intake.

Galacto-oligosaccharides increase calcium absorption and gut bifidobacteria in young girls: a double-blind cross-over trial

Adolescence is a time for rapid growth that represents an opportunity to influence peak bone mass. Prebiotic agents, such as galacto-oligosaccharides (GOS), increase Ca absorption in animal models and postmenopausal women. The objectives of the present study were to investigate the dose-response relationship of GOS supplementation on Ca absorption during growth and to assess changes in colonic microbiota to better understand the mechanism by which GOS is acting. A total of thirty-one healthy adolescent girls aged 10-13 years consumed smoothie drinks twice daily with 0, 2·5 or 5 g GOS for three 3-week periods in a random order. Fractional Ca absorption was determined from urinary Ca excretion over 48 h at the end of each 3-week period using a dual stable isotope method. Faecal microbiota and bifidobacteria were assessed by PCR-denaturing gradient gel electrophoresis and quantitative PCR. Fractional Ca absorption after the 48 h treatment with control, 5 and 10 g GOS/d was 0·393 (SD 0·092), 0·444 (SD 0·086) and 0·419 (SD 0·099), respectively. Significant improvements in Ca absorption were seen with both low and high doses of GOS compared with the control (P,0·02), but itwas not a dose-response relationship. The increase in absorption was greatest in the urine collected after 24 h, which is consistent with lower gut absorption. Faecal bifidobacteria increased (control 10·89 (SD 13·86), 5 g GOS 22·80 (SD 15·74) and 10 g GOS 11·54 (SD 14·20)) with the GOS treatment (P,0·03). The results suggest that daily consumption of 5 g GOS increases Ca absorption, which may be mediated by the gut microbiota, specifically bifidobacteria.

Galactooligosaccharides improve mineral absorption and bone properties in growing rats through gut fermentation.

Galactooligosaccharides (GOS), prebiotic nondigestible oligosaccharides derived from lactose, have the potential for improving mineral balance and bone properties. This study examined the dose-response effect of GOS supplementation on calcium and magnesium absorption, mineral retention, bone properties, and gut microbiota in growing rats. Seventy-five 4-week-old male Sprague-Dawley rats were randomized into one of five treatment groups (n = 15/group) and fed a diet containing 0, 2, 4, 6, or 8% GOS by weight for 8 weeks. Dietary GOS significantly decreased cecal pH and increased cecal wall weight and content weight in a dose-dependent manner (p < 0.0001). Fingerprint patterns of the 16S rRNA gene PCR-DGGE from fecal DNA indicated the variance of bacterial community structure, which was primarily explained by GOS treatments (p = 0.0001). Quantitative PCR of the samples revealed an increase in the relative proportion of bifidobacteria with GOS (p = 0.0001). Net calcium absorption was increased in a dose-response manner (p < 0.01) with GOS supplementation. Dietary GOS also increased (p < 0.02) net magnesium absorption, femur ⁴⁵Ca uptake, calcium and magnesium retention, and femur and tibia breaking strength. Distal femur total and trabecular volumetric bone mineral density (vBMD) and area and proximal tibia vBMD increased (p < 0.02) with GOS supplementation. Trabecular-rich bones, that is, those that rapidly turn over, were most benefited. Regression modeling showed that GOS benefited calcium and magnesium utilization and vBMD through decreased cecal pH, increased cecal wall and content weight, and increased proportion of bifidobacteria.

Randomized Trial of Probiotics and Calcium on Diarrhea and Respiratory Tract Infections in Indonesian Children

OBJECTIVE: To investigate the effects of calcium and probiotics on the incidence and duration of acute diarrhea and acute respiratory tract infections (ARTIs) in low-socioeconomic communities of Jakarta, Indonesia. METHODS: We conducted a 6-month, double-blind, placebo-controlled study in 494 healthy children aged 1 to 6 years who received low-lactose milk with low calcium content (LC; ∼50 mg/day; n = 124), regular calcium content (RC; ∼440 mg/day; n = 126), RC with 5.108 colony-forming units per day of Lactobacillus casei CRL431 (casei; n = 120), or RC with 5.108 colony-forming units per day of Lactobacillus reuteri DSM17938 (reuteri; n = 124). Number and duration of diarrhea and ARTIs episodes were primary and secondary outcomes, respectively. RESULTS: Incidence of World Health Organization–defined diarrhea (≥3 loose/liquid stools in 24 hours) was not significantly different between RC and LC (relative risk [RR]: 0.99 [95% confidence interval (CI): 0.62–1.58]), between casei and RC (RR: 1.21 [95% CI: 0.76–1.92]), or between reuteri and RC (RR: 0.76 [95% CI: 0.46–1.25]) groups. Incidence of all reported diarrhea (≥2 loose/liquid stools in 24 hours) was significantly lower in the reuteri versus RC group (RR: 0.68 [95% CI: 0.46–0.99]). Irrespective of the definition used, reuteri significantly reduced diarrhea incidence in children with lower nutritional status (below-median height-and-weight-for-age z score). None of the interventions affected ARTIs. CONCLUSIONS: RC milk, alone or with L casei, did not reduce diarrhea or ARTIs in Indonesian children. L reuteri may prevent diarrhea, especially in children with lower nutritional status.

Galacto-Oligosaccharides Have Prebiotic Activity in a Dynamic In Vitro Colon Model Using a C-Labeling Technique

Galacto-oligosaccharides (GOS) are considered to be prebiotic, although the contribution of specific members of the microbiota to GOS fermentation and the exact microbial metabolites that are produced upon GOS fermentation are largely unknown. We aimed to determine this using uniformly (13)C-labeled GOS. The normal (control) medium and unlabeled or (13)C-labeled GOS was added to a dynamic, validated, in vitro model of the large-intestine containing an adult-type microbiota. Liquid-chromatography MS was used to measure the incorporation of (13)C label into metabolites. 16S-rRNA stable isotope probing coupled to a phylogenetic micro-array was used to determine label incorporation in microbial biomass. The primary members within the complex microbiota that were directly involved in GOS fermentation were shown to be Bifidobacterium longum, B. bifidum, B. catenulatum, Lactobacillus gasseri, and L. salivarius, in line with the prebiotic effect of GOS, although some other species incorporated (13)C label also. GOS fermentation led to an increase in acetate (+49%) and lactate (+23%) compared with the control. Total organic acid production was 8.50 and 7.52 mmol/g of carbohydrate fed for the GOS and control experiments, respectively. At the same time, the cumulative production of putrefactive metabolites (branched-chain fatty acids and ammonia) was reduced by 55%. Cross-feeding of metabolites from primary GOS fermenters to other members of the microbiota was observed. Our findings support a prebiotic role for GOS and its potential to act as a synbiotic in combination with certain probiotic strains.

CE‐LIF‐MSn profiling of oligosaccharides in human milk and feces of breast‐fed babies

Mixtures of the complex human milk oligosaccharides (HMOs) are difficult to analyze and gastrointestinal bioconversion products of HMOs may complicate analysis even more. Their analysis, therefore, requires the combination of a sensitive and high‐resolution separation technique with a mass identification tool. This study introduces for the first time the hyphenation of CE with an electrospray mass spectrometer, capable to perform multiple MS analysis (ESI‐MSn) for the separation and characterization of HMOs in breast milk and feces of breast‐fed babies. LIF was used for on‐ and off‐line detections. From the overall 47 peaks detected in off‐line CE‐LIF electropherograms, 21 peaks could be unambiguously and 11 peaks could be tentatively assigned. The detailed structural characterization of a novel lacto‐N‐neo‐tetraose isomer and a novel lacto‐N‐fucopentaose isomer was established in baby feces and pointed to gastrointestinal hydrolysis of higher‐Mw HMOs. CE‐LIF‐ESI‐MSn presents, therefore, a useful tool which contributes to an advanced understanding on the fate of individual HMOs during their gastrointestinal passage.

The Effect of Lactulose on the Composition of the Intestinal Microbiota and Short-chain Fatty Acid Production in Human Volunteers and a Computer- controlled Model of the Proximal Large Intestine

The objective of this study was to compare the in vivo effect of lactulose on faecal parameters with the effect in a dynamic, computer-controlled in vitro model of the proximal large intestine (TIM-2). Faecal samples from 10 human volunteers collected before (non-adapted) and after 1 week of treatment (10 g/day) with lactulose (lactulose-adapted) were investigated. Parameters were compared immediately in the faecal samples, and after incubation in the in vitro model of the large intestine. After an adaptation period of the faecal microbiota in the in vitro model of the proximal colon, lactulose (10 g/day) was fed to the microbiota over a 48-h period. Samples taken from the model were investigated for microbiota composition and metabolite production (short-chain fatty acids (SCFAs) and lactate). No changes in the faecal parameters pH, dry weight or SCFA ratio were observed in the in vivo samples. However, the results show a major change in the ratio of SCFAs produced in the in vitro model, with a drastic reduction of butyrate production on lactulose. This was clear in the non-adapted microbiota by the observed arrest in butyrate production 24 h after the start of lactulose feeding. However, in the adapted microbiota butyrate production was already low from the start of the experiment. In fact, only the microbiota of one of the 10 individuals still produced significant amounts of butyrate after lactulose adaptation, the concentration in the other samples was extremely low. Similarly, in the in vitro model lactate production of the non-adapted microbiota started after approximately 24 h, whereas the adapted microbiota produced lactate from the start. In faecal (in vivo) samples no changes in microbiota composition were obvious, except for a significant increase in Bifidobacterium counts after lactulose feeding. With classic plating techniques, the in vitro samples showed an increase in Lactobacillus and Enterococcus species. With denaturing gradient gel electrophoresis, a clear change in banding pattern was observed, indicating a shift in microbiota composition. When the major bands that appeared after lactulose feeding in the in vitro model were excised and sequenced, the sequences showed homology to Lactobacillus and Enterococcus species. This is in agreement with the classic plating technique as well as with the observed increase in lactate production. Sampling in vivo at ‘the site where it all happens’ (the proximal colon) is difficult and inconvenient. We conclude that the in vitro model for the proximal colon reflects much better the fermentation of lactulose, in both metabolite production and changes in microbiota composition, than do faecal samples from an in vivo experiment. Therefore, the in vitro model is an excellent tool with which to study bioconversion of functional food components and/or drugs.

Oligosaccharides in feces of breast- and formula-fed babies

So far, little is known on the fate of oligosaccharides in the colon of breast- and formula-fed babies. Using capillary electrophoresis with laser induced fluorescence detector coupled to a mass spectrometer (CE-LIF-MS(n)), we studied the fecal oligosaccharide profiles of 27 two-month-old breast-, formula- and mixed-fed preterm babies. The interpretation of the complex oligosaccharide profiles was facilitated by beforehand clustering the CE-LIF data points by agglomerative hierarchical clustering (AHC). In the feces of breast-fed babies, characteristic human milk oligosaccharide (HMO) profiles, showing genetic fingerprints known for human milk of secretors and non-secretors, were recognized. Alternatively, advanced degradation and bioconversion of HMOs, resulting in an accumulation of acidic HMOs or HMO bioconversion products was observed. Independent of the prebiotic supplementation of the formula with galactooligosaccharides (GOS) at the level used, similar oligosaccharide profiles of low peak abundance were obtained for formula-fed babies. Feeding influences the presence of diet-related oligosaccharides in baby feces and gastrointestinal adaptation plays an important role herein. Four fecal oligosaccharides, characterized as HexNAc-Hex-Hex, Hex-[Fuc]-HexNAc-Hex, HexNAc-[Fuc]-Hex-Hex and HexNAc-[Fuc]-Hex-HexNAc-Hex-Hex, highlighted an active gastrointestinal metabolization of the feeding-related oligosaccharides. Their presence was linked to the gastrointestinal mucus layer and the blood-group determinant oligosaccharides therein, which are characteristic for the hosts genotype.

Circulating uncarboxylated matrix Gla protein, a marker of vitamin K status, as a risk factor of cardiovascular disease

OBJECTIVES Vitamin K plays a pivotal role in the synthesis of Matrix Gla protein (MGP), a calcification inhibitor in vascular tissue. Vascular calcification has become an important predictor of cardiovascular disease. The aim of the current study was to examine the potential association of circulating desphospho-carboxylated and -uncarboxylated MGP (dp-cMGP and dp-ucMGP), reflecting vitamin K status, with the incidence of cardiovascular events and disease (CVD) in older individuals. STUDY DESIGN The study was conducted in 577 community-dwelling older men and women of the Longitudinal Aging Study Amsterdam (LASA), aged >55 year, who were free of cardiovascular disease at baseline. Multivariate Cox proportional hazards models were used to analyze the data. MAIN OUTCOME MEASURES Incidence of CVD. RESULTS After a mean follow-up of 5.6±1.2 year, we identified 40 incident cases of CVD. After adjustment for classical confounders and vitamin D status, we observed a more than 2-fold significantly higher risk of CVD for the highest tertile of dp-ucMGP with a HR of 2.69 (95% CI, 1.09-6.62) as compared with the lowest tertile. Plasma dp-cMGP was not associated with the risk of CVD. CONCLUSIONS Vitamin K insufficiency, as assessed by high plasma dp-ucMGP concentrations is associated with an increased risk for cardiovascular disease independent of classical risk factors and vitamin D status. Larger epidemiological studies on dp-ucMGP and CVD incidence are needed followed by clinical trials to test whether vitamin K-rich diets will lead to a decreased risk for cardiovascular events.