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Chitin-glucan supplementation improved postprandial metabolism and altered intestine microbiota in topics at cardiometabolic threat in a randomized trial


Moral statements

This examine was reviewed and accredited by the native moral committee CPP Ile de France IV 2018/69, ID-RCB: 2018-A02155-50. It was carried out between November 2018 and June 2019 on the Human Diet Analysis Heart of Rhône-Alpes (CRNH-RA) and carried out in accordance with the Second Declaration of Helsinki and French Jardé’s regulation. It was reported and registered on http://www.clinicaltrials.gov NCT03773900 (12/12/2018). All individuals acquired and signed knowledgeable consent earlier than the initiation of any study-related process.

Research individuals

Sixteen topics with CM threat profile have been recruited. Inclusion standards included age: 30–65 years outdated, BMI: 25–35 kg/m2, waist circumference > 80 cm for ladies and > 94 cm for males, day by day fiber consumption < 25 g/day, exhaled H2 (max–min > 20 ppm) in response to lactulose check, steady weight and average bodily exercise, no recognized gastrointestinal illness, no beforehand bariatric surgical procedure, no use of antibiotics or different medication interfering with microbiota composition within the 3 months previous to the start of the examine.

Research design

This was a dietary monocentric, randomized, double blind and cross-over examine. Every topic acquired two 3-week dietary interventions in a random order in response to a randomization record per block of permutations established with SAS software program 9.4 by the statisticians. The 2 dietary interventions have been, 1) 4.5 g/d of chitin-glucan (CG) and a pair of) 4.5 g of maltodextrin as management (Desk S3). A 4 to 6-week washout interval separated the 2 interventions. Earlier than and after every intervention, fecal samples have been collected for intestine microbiota composition and its derived-metabolites ‘evaluation. Fasting and postprandial metabolic parameters have been evaluated throughout metabolic evaluation days (Determine S3).

CG and CTL had the identical galenic kind and visible facet however primarily differed by their fiber content material (Desk S3). Each have been offered as a 3 g sachet powder to be diluted in water. Every sachet included 1.5 g of CG or 1.5 g management, thus topics consumed 3 sachets per day (20–30 min earlier than the three meals of the day) to achieve the goal dose of 4.5 g/d. The set of sachets to be consumed for every interval was given to topics originally of every interval throughout which, they have been requested to maintain the identical eating regimen and regular bodily exercise. A French Nationwide Authority for Well being (HAS) questionnaire assessed with NUTRILOG software program (model 3.10b, launched in February 2017) was used to judge topics’ dietary fiber consumption at inclusion. To judge dietary consumption and guarantee an excellent compliance throughout the intervention intervals, topics have been ordered to fill in a 3-day dietary file. Information from this 3-day dietary file have been additionally processed utilizing NUTRILOG software program which allowed to estimate power, macronutrient, and dietary fiber intakes. Research’s merchandise consumption compliance was evaluated by counting full and empty sachets they dropped at the middle on the finish of each intervention interval.

Through the 3 days earlier than metabolic evaluation day, topics have been ordered to gather stool samples utilizing a particular equipment. Stool samples have been saved within the topic’s freezer till they introduced them to the middle utilizing a furnished cooler bag. On metabolic evaluation days, topics arrived at CRNH-RA after an 8-h in a single day quick following the ingestion of a standardized low dietary fiber night meal (one serving of fish, rice, cheese, rusk or smooth bread, dried biscuits and butter). Physique weight and fats mass share have been measured utilizing standardized methodologies utilizing a calibrated weighing scale and a Bodystat Quadscan 4000 (BQ4000; Bodystat Ltd. Douglas, UK) respectively. RMR was measured by oblique calorimetry utilizing a QUARK calorimeter (Cosmed, Rome, Italy). Topics have been served a dietary fiber-enriched breakfast at T0 (rye bread, hazelnut carob unfold, fruit juice) and a standardized problem check meal: the FlexMeal problem at T240. The FlexMeal is a revised model of the “PhenFlex drink”43. It consists on a standardized problem check meal composed by a soup and a dairy dessert presenting 923 kcal, 32.5% of carbohydrates, 58.9% of lipids and eight.7% of proteins. Fasting and 4 h postprandial blood (T0, T30, T60, T90, T120, T180, T240, T255, T270, T300, T330, T360, T420) have been collected utilizing an antecubital vein catheter. Fasting and postprandial exhaled gases have been additionally collected (T0, T60, T120, T180, T240, T300, T360, T420, T480, T540, T600).

Biochemical blood analyses

Collected blood was centrifuged instantly for 10 min at 4500 rpm. Plasma was saved at − 20 °C till the assays have been carried out.

Glycemia was measured by spectrophotometry in response to Architect Abbott Hexokinase methodology; TC, HDL-C and TG by spectrometry utilizing Architect Module Chimie Abbott methodology; insulin by radio immunoassay in response to RIA CisBio IBA methodology, NEFA by automatized spectrometry with PENTRA 400 HORIBA ABX. LDL-C was calculated utilizing Friedwald method and HOMA as plasma glucose (mmol/L) x plasma insulin (mUI/L) / 22.5.

Exhaled gases analyses

Exhaled gases have been collected utilizing EASYSAMPLER Breath Take a look at Package (Quintron, Milwaukee, WI, USA). The concentrations of H2, CH4 and CO2 have been then measured by gasoline chromatography utilizing a QUINTRON BREATH TRACKER analyzer (Quintron, Milwaukee, WI, USA).

Microbiota evaluation

Fecal microbiome sequencing evaluation

Bacterial DNA was extracted from fecal samples utilizing the QIAamp DNA Stool Mini Package (QIAGEN, Hilden, Germany), following the ‘Protocol Q’ that was described by Costea and colleagues44 with a slight modification: a discount in time of bead beating step. Cells are mechanically lysed by operating the FASTPREP Instrument for two min at max velocity (beating 1 min and resting 5 min).

Library building and the Illumina sequencing protocol has been beforehand described intimately45. Briefly, the V5-V6 areas of the 16S rRNA gene have been focused for PCR amplification utilizing primers 784 F [5′-RGGATTAGATACCC-3′] and 1064 R [5′- CGACRRCCATGCANCACCT-3′]. 16S rRNA gene amplicons have been sequenced by the MiSeq platform (300 bp paired-end size) on the College of Minnesota Genomics Heart. All samples of this examine have been sequenced in the identical run. Paired-end reads have been merged, demultiplexed and carried out high quality management implementation (size = 281 bp, imply sequence high quality rating ≥ 30) utilizing QIIME2 pipeline with DADA246,47, which we discuss with herein as amplicon sequences variants (ASV). The common of the sequence in all samples (n = 60) was 19,884. An excellent depth of 8,290 sequences per pattern was used to conduct microbiome range. We assigned the sequences to taxonomic classes together with kingdom, phylum, class, order, household and genus ranges utilizing a pre-trained Naive Bayes classifier based mostly on Silva 132 99% OTUs database48. For vital ASV, with a view to have a better decision of the ASV identification, on-line 16S rRNA databases on each NCBI blast and EzBioCloud platforms have been additionally used.

SCFA evaluation

The methodology has been described earlier than15. Briefly, the native fecal samples have been homogenized and subsamples of 400–500 mg have been diluted 1:4 in ultrapure water and analyzed utilizing a capillary gasoline chromatograph (HP6890 Sequence; Hewlett Packard Corp., Paolo Alto, California, USA). Fecal dry mass was assessed by drying 300–500 mg of native pattern in a single day at 103 °C.

Markers of intestinal permeability

All biomarkers included have been validated as markers of intestinal permeability earlier than (https://pubmed.ncbi.nlm.nih.gov/34009040/). Zonulin and calprotectin have been measured utilizing enzyme-linked immunosorbent assay kits (K5600; K6927; Immundiagnostik AG, Bensheim, Germany) following the producer’s protocol. The fecal samples have been diluted to the working focus in pattern buffer utilizing stool pattern tubes (K6998SAS; Immundiagnostik AG, Bensheim, Germany). LBP (REFs: DY870-05 and DY008; Bio-Techne GmbH, Wiesbaden, Germany) was measured in plasma.

Bile acids evaluation

Bile acids have been analyzed utilizing methodology validated methodology tailored from Guillemot-Legris et al.49. Briefly, lyophilized feces (5 mg) have been homogenized in ice-cold distilled water previous to protein precipitation utilizing acetone containing seven deuterated bile acids used as inner requirements. Samples have been subsequent centrifuged, and the supernatant was evaporated to dryness underneath nitrogen steam. The ensuing residue was resuspended in methanol and injected within the HPLC–MS system consisting of an LTQ-Orbitrap XL mass spectrometer (Thermo Fisher Scientific) coupled to an Accela HPLC system (Thermo Fisher Scientific). Analyte separation was carried out on an Ascentis Specific C-18 column (2.7 µm, 100 × 4.6 mm) (Sigma-Aldrich) utilizing a gradient between acetonitrile and water, each containing formic acid. Mass spectrometry evaluation was carried out utilizing an electrospray ionization supply within the unfavourable mode. Calibration curves have been ready in the identical circumstances.

LCFA evaluation

To find out the LCFA profile in feces, we used 40 mg of beforehand lyophilized feces throughout 48 h (Labconco, freeze dryer 4.5). Forty microliter of C19 have been added as extraction customary for homogenization with methanol:chloroform (1:2 V/V) by transient sonication in ice (Labsonic U, B. Braun). Homogenates have been then filtered with Whatman filters n°1 (10 μm of porosity). Filters have been rinsed with 2 ml of chloroform and 1 ml of methanol. Homogenates have been purified with KCl 0.88% and KCl 0.88%: methanol (1:1 V/V). After centrifugation (1500 g, 5 min), the chloroform section was collected in new tubes and evaporated underneath nitrogen flux till samples have been fully dry. The esterified fatty acids have been then subjected to alkaline hydrolysis (saponification) and free fatty acids have been methylated and quantified by gasoline chromatography with flame ionization detector as beforehand described50.

Statistics

The first final result of this examine was the measure of SCFA manufacturing. As a result of lack of obtainable revealed information on this particular fiber, an ordinary energy calculation to acquire the variety of topics wanted for the examine was not attainable. The selection of the first final result relayed on information from two research. The primary one, carried out in an in vitro mannequin simulating the gut confirmed {that a} dose of 4.5 g of CG was capable of improve the manufacturing of SCFA13. The second confirmed {that a} supplementation of three g/day for two months of BG, a part of CG and a dietary fiber which construction is carefully associated CG, resulted in a major improve of SCFA within the feces of 26 topics51.

All statistical analyses and graphs have been carried out with R software program v3.6.0 and SAS software program 9.4 TS Degree 1M652.

To seek out out whether or not CG had a distinct impact from the impact of management on scientific variables and to evaluate if the distinction was statistically vital, we carried out a linear combined mannequin for repeated measures, with Toeplitz or autoregressive construction (AR) as covariance construction. With the intention to account for variability between topics and to regulate for any non-specific variations, topics have been included as random results. To quantify the impact of CG in comparison with the impact of management, we estimated the distinction by way of change ETD = After Fiber − Earlier than Fiber − (After Management − Earlier than Management) to which the p worth of the interplay remedy*time is related.

For intestine microbiota evaluation, relative abundances carried out in Qiime2 have been calculated in R for every taxa and ASV. To keep away from analyzing spurious sequences, ASV with a median relative abundance under 0.1% in all samples have been eliminated. The identical cut-off was utilized for evaluation of bacterial genera. To evaluate if a given microbial characteristic was otherwise impacted by the 2 remedies, we first computed the delta (after–earlier than) in abundance for each remedies. Then, a Wilcoxon signed-rank check has been carried out to evaluate if the distinction between the 2 deltas was statistically vital; in that case, we evaluated the affect of every remedy by evaluating the abundances earlier than and after its consumption with a Wilcoxon signed-rank check.

Beta-diversity indices have been in contrast by PERMANOVA utilizing the Adonis perform within the vegan bundle on R Software program and information have been visualized utilizing non-metric multidimensional scaling.

For subgroup evaluation, we computed Δ exhaled H2 = exhaled H2 peak – baseline and outlined two subgroups: “Excessive-H2” = topics with Δ exhaled H2 > Δ exhaled H2 median and “Low-H2” = topics with Δ exhaled H2 < Δ exhaled H2 median. The interplay remedy*time*subgroup was examined for all variables and the linear combined mannequin for repeated measures was solely carried out when the interplay was vital (p < 0.05).

For correlation analyses, we computed the deltas (after–earlier than) following CG supplementation for every metabolic parameter and bacterial characteristic which have been otherwise impacted by the 2 intervention. Spearman’s correlations have been carried out.

All statistical analyses have been carried out with adjustment utilizing false discovery charge (FDR < 0.05) for a number of exams in response to the Benjamini–Hochberg process and not one of the adjusted p values have been vital. However, non-adjusted p values < 0.05 have been thought-about vital because of the explorative character of those approaches.

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