Henrik Lauritz Frandsen

Colonic transit time is related to bacterial metabolism and mucosal turnover in the gut

Little is known about how colonic transit time relates to human colonic metabolism and its importance for host health, although a firm stool consistency, a proxy for a long colonic transit time, has recently been positively associated with gut microbial richness. Here, we show that colonic transit time in humans, assessed using radio-opaque markers, is associated with overall gut microbial composition, diversity and metabolism. We find that a long colonic transit time associates with high microbial richness and is accompanied by a shift in colonic metabolism from carbohydrate fermentation to protein catabolism as reflected by higher urinary levels of potentially deleterious protein-derived metabolites. Additionally, shorter colonic transit time correlates with metabolites possibly reflecting increased renewal of the colonic mucosa. Together, this suggests that a high gut microbial richness does not per se imply a healthy gut microbial ecosystem and points at colonic transit time as a highly important factor to consider in microbiome and metabolomics studies.

Effect of a long-chained fructan Raftiline HP on blood lipids and spontaneous atherosclerosis in low density receptor knockout mice

Abstract The effect of a long-chained fructan Raftiline HP on spontaneous hypercholesterolemia and atherosclerosis was studied in 40 LDLR -/- male mice receiving isocaloric, balanced in fat content, purified diets with 0 or 10% Raftiline HP, for 16 weeks. The feed intake was comparable (3.9 v. 3.8 g/day) but the terminal body weight was lower in the Raftiline HP group (36 v. 32g, p v. 6.41 mmol/l, at termination) and its concentrations in LDL (2.36 v. 1.7 mmol/l), IDL (1.27 v. 0.77 mmol/l) and VLDL (1.53 v. 0.96 mmol/l) but not in HDL (1.13 v. 1.11). Plasma triglycerides were comparable between the groups (1.14 v. 1.04 mmol/l, at termination). The ratio intima/media was 15% lower (0.089 v. 0.076), although not statistically significantly different. In conclusion, the findings of this study emphasize the cholesterol lowering potential of inulin.

Authenticity and Traceability of Vanilla Flavors by Analysis of Stable Isotopes of Carbon and Hydrogen

Authenticity and traceability of vanilla flavors were investigated using gas chromatography-isotope ratio mass spectrometry (GC-IRMS). Vanilla flavors produced by chemical synthesis (n = 2), fermentation (n = 1), and extracted from two different species of the vanilla orchid (n = 79) were analyzed. The authenticity of the flavor compound vanillin was evaluated on the basis of measurements of ratios of carbon stable isotopes (δ(13)C). It was found that results of δ(13)C for vanillin extracted from Vanilla planifolia and Vanilla tahitensis were significantly different (t test) and that it was possible to differentiate these two groups of natural vanillin from vanillin produced otherwise. Vanilla flavors were also analyzed for ratios of hydrogen stable isotopes (δ(2)H). A graphic representation of δ(13)C versus δ(2)H revealed that vanillin extracted from pods grown in adjacent geographic origins grouped together. Accordingly, values of δ(13)C and δ(2)H can be used for studies of authenticity and traceability of vanilla flavors.

Simultaneous quantification of vitamin D3, 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 in human serum by LC-MS/MS

Abstract Introduction. Serum 25-hydroxy-vitamin D is the established biomarker of vitamin D status although serum concentrations of vitamin D and 24,25-dihydroxyvitamin D may also be of interest to understand the in vivo kinetics of serum 25-hydroxyvitamin D. Method. An LC-MS/MS method was developed and validated to quantify vitamin D3, 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 in serum. After protein precipitation of the serum it was loaded on a HybridSPE column to separate vitamin D metabolites from phospholipids. Vitamin D3, 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 in the eluate were derivatized by 4-phenyl-1,2,4-triazoline-3,5-dione to improve sensitivity in the following LC-MS/MS analysis. Results. Using only 100 μL serum the limit of quantification was < 0.2 ng/mL for vitamin D3, 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3. The method was validated up to 100 ng/mL (260 nmol/L) for vitamin D3, up to 100 ng/mL (240 nmol/L) for 24,25-dihydroxyvitamin D3 and up to 200 ng/mL (499 nmol/L) for 25-hydroxyvitamin D3. Precision was < 6.5% for vitamin D3 and 25-hydroxyvitamin D3 and < 10.2% for 24,25-dihydroxyvitamin D3. Conclusion. We demonstrate that a method including not only serum 25-hydroxyvitamin D3 but also vitamin D3 and 24,25-dihydroxyvitamin D3 could easily be implemented in most modern biochemical laboratories. The method could be used to study the metabolism of endogenous synthesized vitamin D3 as well as vitamin D3 in intervention studies.

Microbiotas from UC patients display altered metabolism and reduced ability of LAB to colonize mucus

We compared fecal microbial communities derived either from Ulcerative Colitis (UC) patients in remission (n = 4) or in relapse (n = 4), or from healthy subjects (n = 4). These communities were used for inoculation of a dynamic in vitro gut model, which contained integrated mucin-covered microcosms. We found that the microbiota of the ‘mucus’ largely differed from that of the ‘lumen’. This was partly due to decreased mucus-associated populations of lactic acid producing bacterial populations (LAB), as LAB originating from UC patients had a significantly decreased capacity to colonize the mucin-covered microcosms as compared to those originating from healthy subjects. We found significant differences between the metabolomes of UC patients in relapse and remission, respectively, while the metabolome of patients in remission resembled that of healthy subjects. These novel findings constitute an important contribution to the understanding of the complex etiology of UC.

Whole grain-rich diet reduces body weight and systemic low-grade inflammation without inducing major changes of the gut microbiome: a randomised cross-over trial

Objective To investigate whether a whole grain diet alters the gut microbiome and insulin sensitivity, as well as biomarkers of metabolic health and gut functionality. Design 60 Danish adults at risk of developing metabolic syndrome were included in a randomised cross-over trial with two 8-week dietary intervention periods comprising whole grain diet and refined grain diet, separated by a washout period of ≥6 weeks. The response to the interventions on the gut microbiome composition and insulin sensitivity as well on measures of glucose and lipid metabolism, gut functionality, inflammatory markers, anthropometry and urine metabolomics were assessed. Results 50 participants completed both periods with a whole grain intake of 179±50 g/day and 13±10 g/day in the whole grain and refined grain period, respectively. Compliance was confirmed by a difference in plasma alkylresorcinols (p<0.0001). Compared with refined grain, whole grain did not significantly alter glucose homeostasis and did not induce major changes in the faecal microbiome. Also, breath hydrogen levels, plasma short-chain fatty acids, intestinal integrity and intestinal transit time were not affected. The whole grain diet did, however, compared with the refined grain diet, decrease body weight (p<0.0001), serum inflammatory markers, interleukin (IL)-6 (p=0.009) and C-reactive protein (p=0.003). The reduction in body weight was consistent with a reduction in energy intake, and IL-6 reduction was associated with the amount of whole grain consumed, in particular with intake of rye. Conclusion Compared with refined grain diet, whole grain diet did not alter insulin sensitivity and gut microbiome but reduced body weight and systemic low-grade inflammation. Trial registration number NCT01731366; Results.

Quantification of physiological levels of vitamin D3 and 25‐hydroxyvitamin D3 in porcine fat and liver in subgram sample sizes

Most methods for the quantification of physiological levels of vitamin D3 and 25-hydroxyvitamin D3 are developed for food analysis where the sample size is not usually a critical parameter. In contrast, in life science studies sample sizes are often limited. A very sensitive liquid chromatography with tandem mass spectrometry method was developed to quantify vitamin D3 and 25-hydroxyvitamin D3 simultaneously in porcine tissues. A sample of 0.2-1 g was saponified followed by liquid-liquid extraction and normal-phase solid-phase extraction. The analytes were derivatized with 4-phenyl-1,2,4-triazoline-3,5-dione to improve the ionization efficiency by electrospray ionization. The method was validated in porcine liver and adipose tissue, and the accuracy was determined to be 72-97% for vitamin D3 and 91-124% for 25-hydroxyvitamin D3 . The limit of quantification was <0.1 ng/g, and the precision varied between 1.4 and 16% depending on the level of spiking. The small sample size required for the described method enables quantification of vitamin D3 and 25-hydroxyvitamin D3 in tissues from studies where sample sizes are limited.

Tissue content of vitamin D3 and 25-hydroxy vitamin D3 in minipigs after cutaneous synthesis, supplementation and deprivation of vitamin D3

Information regarding the endogenous storages of vitamin D3 after cutaneous vitamin D synthesis compared to oral vitamin D3 supplementation is sparse. Furthermore it is not known whether vitamin D3 can be stored for later use during periods of shortages of vitamin D3. To investigate the endogenous storages of vitamin D3 two studies were carried out in Göttingen minipigs. In study 1 one group of minipigs (n=2) was daily exposed to UV light corresponding to 10-20 min of midday sun and another group (n=2) of pigs were fed up to 60 μg vitamin D3/day corresponding to 3.7-4.4 μg/kg body weight. Study 1 demonstrated that daily UV-exposure of minipigs stimulated the cutaneous synthesis of vitamin D3 and resulted in increasing serum vitamin D3 and 25-hydroxy vitamin D3, but also carcasses containing vitamin D3 and 25-hydroxy vitamin D3. The vitamin D3 content in adipose tissue from the UV-exposed minipigs was 150-260 ng/g and the content was 90-150 ng/g in the orally supplemented minipigs. In study 2, minipigs were UV-exposed daily for 49 days. Subsequently, one group (n=2) was fed a vitamin D-free diet and another group (n=2) was dosed daily with 13C-labeled vitamin D3. The concentrations of vitamin D3 and 25-hydroxy vitamin D3 in serum and skin- and subcutaneous adipose tissue biopsies were repeatedly monitored. Vitamin D3 and 25-hydroxy vitamin D3 were eliminated from the skin and the adipose tissue after UV-exposure was ceased. Supplementation of 13C-vitamin D3 did not seem to affect the decline in the endogenous vitamin D3 in the adipose tissue formed during UV-exposure.

Environmental spread of microbes impacts the development of metabolic phenotypes in mice transplanted with microbial communities from humans.

Microbiota transplantation to germ-free animals is a powerful method to study involvement of gut microbes in the aetiology of metabolic syndrome. Owing to large interpersonal variability in gut microbiota, studies with broad coverage of donors are needed to elucidate the establishment of human-derived microbiotas in mice, factors affecting this process and resulting impact on metabolic health. We thus transplanted faecal microbiotas from humans (16 obese and 16 controls) separately into 64 germ-free Swiss Webster mice caged in pairs within four isolators, with two isolators assigned to each phenotype, thereby allowing us to explore the extent of microbial spread between cages in a well-controlled environment. Despite high group-wise similarity between obese and control human microbiotas, transplanted mice in the four isolators developed distinct gut bacterial composition and activity, body mass gain, and insulin resistance. Spread of microbes between cages within isolators interacted with establishment of the transplanted microbiotas in mice, and contributed to the transmission of metabolic phenotypes. Our findings highlight the impact of donor variability and reveal that inter-individual spread of microbes contributes to the development of metabolic traits. This is of major importance for design of animal studies, and indicates that environmental transfer of microbes between individuals may affect host metabolic traits.

LC-MS analysis of the plasma metabolome–a novel sample preparation strategy

Blood plasma is a well-known body fluid often analyzed in studies on the effects of toxic compounds as physiological or chemical induced changes in the mammalian body are reflected in the plasma metabolome. Sample preparation prior to LC-MS based analysis of the plasma metabolome is a challenge as plasma contains compounds with very different properties. Besides, proteins, which usually are precipitated with organic solvent, phospholipids, are known to cause ion suppression in electrospray mass spectrometry. We have compared two different sample preparation techniques prior to LC-qTOF analysis of plasma samples: the first is protein precipitation; the second is protein precipitation followed by solid phase extraction with sub-fractionation into three sub-samples: a phospholipid, a lipid and a polar sub-fraction. Molecular feature extraction of the data files from LC-qTOF analysis of the samples revealed 1792 molecular features from the protein precipitation procedure. The protein precipitation followed by solid phase extraction procedure with three sub-samples gave a total of 4234 molecular features. This suggests that sub-sampling into polar, lipid and phospholipid fractions enables extraction of more metabolomic information as compared to protein precipitation alone. Chromatography showed good separation of the metabolites with little retention time drift (<1s) and a mass accuracy below 3 ppm was observed. The performance of the method was investigated using plasma samples from rats administered the environmental pollutant perfluorononanoic acid.