A. Rehman et al.
The Journal of Nutrition xxx (xxxx) xxx
TMAO and LPS samples were sent to MS-Omics ApS (Bygstubben 9, Vedbaek, Denmark) for analysis using a Ultra Performance Liquid Chromatography (UPLC) system (Vanquish, Thermo Fisher Scienti fi c) coupled with a high resolution quadrupole-orbitrap mass spectrometer (Q Exactive HF Hybrid Quadrupole-Orbitrap, Thermo Fisher Scienti fi c). Plasma BA were extracted from plasma and quanti fi edusinga commercially available BA assay (Biocrates Life Sciences AG). SCFAs and other organic acids were fi rst extracted from the plasma by protein precipitation. Then plasma extracts were derivatized and reaction products extracted by liquid-liquid extraction using dichloromethane. Obtained extracts were fi nally injected into a Ultra High Performance Liquid Chroma- tography (UHPLC)-MS/MS system for analysis in combined positive and ESI MRM mode. For untargeted metabolomics, plasma samples were prepared using a previously described method with minor adaptations [26]. In short, 100 μ L of human plasma was mixed with 100 μ Lof a deuterated phosphate buffer pH ¼ 7.3, prepared by adding a Phosphate Buffered Saline (PBS) tablet (OXOID) in 100 mL of D 2 O. Maleic acid (0.5mM) was used as an internal standard, and the samples were subsequently put in 3-mm Nuclear Magnetic Resonance (NMR) tubes. All spectra were acquired at 298 K on a Bruker Avance III NMR spectrometer operating at 600 MHz proton Larmor frequency and equipped with a 5 mm triple resonance inverse cryoprobe. 1D 1 H NMR spectra were acquired using a cpmgpr1d pulse sequence, relaxation and D1 and D20 delays of 5 and 0.0003 seconds respectively; 256 scans were accumulated in 36.5 minutes per spectrum. Fecal samples Fecal samples were collected in DNA/RNA Shield and fecal collection tubes (Zymo Research) and delivered on dry ice in 80 º C compatible boxes to BaseClear BV for microbiome pro fi ling. In brief, nucleic acid was extracted from fecal samples using the ZymoBIOMICS DNA Miniprep (Zymo Research Corp.) kit per manufacturer s instruction. 16S rRNA gene variable region V3- V4 was ampli fi ed by composite primer 341F (5 ’ - CCTACGGGNGGCWGCAG-3') and 785R (5 ’ -GACTACHVGGG- TATCTAATCC – 3') and sequenced using the Illumina MiSeq sequencing platform to generate paired-end sequence reads. Subsequently, reads containing the PhiX control signal were removed by aligning the sequence reads against the PhiX genome (NC_001422.1) with Bowtie2 (2.2.6) [27] and removed from further analysis. In addition, reads containing (partial) adapters were clipped (up to a minimum read length of 50 bp). The second quality assessment was based on the remaining reads using the FASTQC quality control tool version 0.11.5. Paired-end sequence reads were collapsed into the so-called pseudoreads using sequence overlap with USEARCH version 9.2 [28]. Classi fi cation of these pseudoreads is performed based on the results of align- ment with SNAP version 1.0.23 [29] against the RDP database [30] for bacterial organisms. Alpha diversity indices (observed species, Chao1, Shannon and Simpson diversity indices) and beta diversity indices (Jaccard and Bray – Curtis) were calculated by implementing mothur version 1.35.1 [31]. Stool consistency and gastrointestinal symptoms Stool consistency was assessed using the Bristol Stool Chart: Type 1 (separate hard lumps, like nuts), Type 2 (sausage-shaped
Participants were provided with another stool collection kit as well as Bristol Stool Chart and instructed to collect a stool sample at home, within 24 hours of their next scheduled visit at week 4. At visit 3, participants again arrived at the study site, fasted overnight, and returned the stool samples and Bristol Stool Chart. Another blood and urine sample was collected, and a Gastroin- testinal Symptom Rating Scale was completed. In addition, par- ticipants returned any unused study product to assess compliance with the intervention phase 1. Participants were then sent home for the 6-week wash-out before entering the intervention phase 2, following the same experimental setup as visits 2 and 3. Subgroup egg challenge To determine the effect of the intervention on postprandial plasma TMAO concentrations, a subgroup of 9 participants completed an acute egg challenge at visits 3 and 5, respectively. For this, participants were instructed to follow a standardized menu, designed to deliver a low-choline diet for the 24-hours before the study visits. On the day of the experiments, partici- pants arrived in the morning and fasted overnight, and a urine sample was collected. Subsequently, an intravenous cannula was placed in a forearm vein, and a baseline blood sample (T0) was collected. Participants then consumed the egg breakfast within 10 minutes, and additional blood samples (6 mL) were drawn for the measurement of plasma TMAO at 120, 240, 360, and 480 minutes postprandially. In addition, all urine samples were collected over an 8-hour period and analyzed for TMAO. Par- ticipants were required to stay at the study site during this time and were provided with a standardized lunch and snacks. The egg breakfast consisted of a high-yolk scrambled egg recipe using pasteurized liquid egg yolks and egg whites to control the vol- ume of egg yolk. The total choline content of the egg breakfast was approximately 450 mg. The provided lunch and snacks were of fi xed calorie content and low in total choline. Participants were advised to fi nish each provided meal. Ethics statement The trial was conducted by Atlantia Food Clinical Trials Ltd., in Cork, Ireland in accordance with the Declaration of Helsinki and approved by the Clinical Research Ethics Committee of the Cork Teaching Hospitals. Written informed consent for partici- pation was obtained from all participants. The trial was regis- tered with clinicaltrials.gov (NCT04160481).
Measurements Blood and urine samples
Blood samples were collected using EDTA plasma tube for safety pro fi ling (hematology, chemistry, glucose, and bilirubin) and analysis of TMAO, LPSs, BAs, SCFAs, and other organic acids as well as untargeted metabolomics. In addition, a serum sample was collected for analysing the levels of high-sensitivity CRP (hsCRP) and urine samples for the analysis of urine TMAO. Safety pro fi ling, including hematology, chemistry, and glucose, was performed by standard clinical laboratory methods in Euro fi ns Biomnis. Bilirubin and hsCRP samples were sent to Euro fi ns Biomnis for analysis as per Biomins standard proced- ures. Bilirubin was assessed using a diazo reaction assessment to determine the total bilirubin and direct bilirubin values, and hsCRP was assessed via immunoturbidimetric determination using a Multigent CRP Vario assay.
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