Monkey (Macaca fascicularis) samples (plasma, urine, and stool) from 20 male animals (aged 3 years 10 months to 6 years 5 months), housed for more than 8 months under the same conditions, were obtained from LSI Medience Co. (Tokyo, Japan) after review by the Animal Experimentation Committee, and approval by the Director of the Testing and Research Center in LSI Medience Co. (Approval No. 2018 − 1071). The monkeys were housed with the lighting turned on from 7:00 to 19:00 and turned off at night. The cages were cleaned once a day. The monkeys were fed 100 g of solid feed (CLEA Old World Monkey Diet CMK-2, CLEA Japan Inc., Tokyo) once a day. Filtered (5 µm filter) and UV irradiated tap water was provided as drinking water. Monkeys have a habit of urinating when the lights are turned on in the morning, so urine was collected early in the morning by placing a urine collection tray in the home cage and lighting up the animal room. Regarding stool collection, since we did not know when the monkeys would defecate, a tray for feces was placed in the home cage, and the animal caretakers collected the stool into a plastic tube as soon as they found one. On the same day as urine collection, approximately 5 mL blood was drawn in a tube with EDTA-2K and centrifuged (1750 x g, 10 min, 4°C) to obtain approximately 2 mL plasma. All samples were frozen and stored in the − 80°C freezer at LSI Medience immediately after collection and kept on dry ice during transport.
To carry out targeted lipidomics/fatty acid analysis by Gas Chromatography with Flame Ionization Detection (GC-FID), 50 µL plasma samples and 20 mg wet weight of feces and feed (The feces and feed were dissolved while dipped in the sonication bath, and the feed was fully pulverized with a stainless-steel crusher) were derivatized using the Fatty Acid Methylation Kit (Nacalai Tesque, Tokyo) with C23:0 fatty acid solution (n-Tricosanoic acid, Sigma-Aldrich) as an internal standard.
For untargeted lipidomics by LC/MS, 20 µL of plasma was combined with 180 µL of methanol, vortexed, and centrifuged to collect the supernatant. The supernatant was further diluted three times with methanol for the analysis. Urine lipids were recovered from the organic layer of 300 µL urine using the Bligh & Dyer method, followed by evaporation of the organic solvent, and resuspension of the residuals in 60 µL methanol for measurement. About 20 mg of feces was weighed (wet weight) and mixed with 20 µL water, and kept on ice. After vortexing, 180 µL methanol was added, and the mixture was stirred in an ultrasonic bath with ice cooling. After centrifugation, the supernatant was diluted four times with methanol and used to measure phospholipids. Twenty milligrams of feed was ground and extracted with 500 µL methanol. After centrifugation, the supernatant was collected and diluted with methanol to 5 mg/mL.
For targeted lipidomics by LC/MS, solid-phase extraction (SPE) was performed as described previously with some modifications54. 200 µL of plasma was combined with 0.8 mL of methanol and mixed with internal standards (18 heavy water labelled components) and centrifuged to collect the supernatant. The supernatant was then purified using an Oasis HLB cartridge (10mg, Waters, Milford, MA) and eluted with 200 µL of 0.2% formic acid in methanol. The solvent was evaporated by rotary evaporator and resuspended in 50 µL of methanol for measurement. Four hundred µL of urine was centrifuged after mixing with 800 µL of methanol and internal standards, and the supernatant was applied to Oasis HLB cartridge, as mentioned above for plasma. The eluate was measured. About 150 mg of feces was measured as wet weight, and 300 µL of water was added on ice and mixed well. Subsequently, 1.2 mL of methanol and internal standards were added, mixed in an ultrasonic bath while chilled/ The supernatant was purified using an Oasis HLB cartridge as mentioned above for plasma samples, and eluted with 200 µL of 0.2% formic acid in methanol. The eluate was analyzed by LC/MS.
To carry out untargeted metabolomics for hydrophilic metabolites analysis by LC/MS, 560 µL methanol was added to 140 µL plasma and vortexed, centrifuged, and 600 µL supernatant was concentrated in a rotary evaporator and resuspended in 100 µL methanol for measurement. For urine, the upper layer (aqueous phase) of the Bligh & Dyer extraction with 300 uL urine was dried up by a rotary evaporator and resuspended in 100 µL methanol for measurement. For feces, about 20 mg wet weight of feces was suspended in 20 µL water on ice, followed by the addition of 180 µL methanol, and mixed in an ultrasonic bath while chilled. After centrifugation, the supernatant was diluted four times with methanol and measured. The 20 mg food was smashed, mixed well with 800 µL methanol, centrifuged to collect the supernatant, and then diluted 10-fold with methanol for measurement.
For trace element measurements, each sample (50 mg for feed samples, 50 µL each for plasma samples, 50 µL each for urine samples, and the entire volume of each feces sample) was placed into a 50-mL tall beaker and heated on hotplates to 150°C. After 3 minutes, 2 mL 60% (v/v) nitric acid (HNO3 for poisonous metal determination; Kanto Chemical, Tokyo, Japan) was added, followed 3 minutes later by 2 mL 60% (v/v) perchloric acid (HClO4 for poisonous metal determination; Kishida Chemical, Osaka, Japan), and 3 minutes later by 2 mL 30% (v/v) hydrogen peroxide (H2O2 for atomic absorption spectrochemical analysis; Kishida Chemical, Osaka, Japan). This process was repeated three times, and if any brownish residue remained, it was repeated further until the sample was completely white. Thus, ashing was continued until the residual material at the bottom of the tall beaker turned completely white. After the sample was cooled to room temperature, 5 mL (feed, plasma, and urine) or 10 mL (feces) of 5% (v/v) nitric acid was added and allowed to stand at room temperature for 24 hours to completely dissolve any remaining material. Then, 5 µL (feed, plasma, and urine) or 10 µL (feces) of 1 µg/mL indium (In) was added as an internal standard solution, mixed well, and each sample solution was transferred to a polycarbonate cup for ICP-MS measurement. Tall beakers and sample cups were previously soaked in 1% nitric acid for at least 3 days, washed with ultrapure water, and dried.
For 16S rRNA gene amplicon library construction and sequencing, extraction of DNA from fecal samples (approximately 200 mg of biomass per sample) was performed using the ISOSPIN Fecal DNA kit (Nippon Gene Co., Ltd.), according to manufacturer’s instructions. For cell lysis, three rounds (1 min each) of bead beating were performed using the FastPrep-24 instrument (MP Biomedicals) at a speed of 6 m/s. DNA concentrations were measured with the Quant-iT PicoGreen dsDNA Assay Kit using a Qubit fluorometer (both from Invitrogen). Amplicon sequencing libraries were prepared by two-step tailed polymerase chain reaction (PCR) following Illumina’s “16S Metagenomic Sequencing Library Preparation” protocol. In the first round of PCRs, the V4 hypervariable region of the 16S rRNA gene was amplified using primers 515F (5′-GTGYCAGCMGCCGCGGTAA-3′) 55 and 806R (5′-GGACTACNVGGGTWTCTAAT-3′)56; primers contained appropriate 5′-end adapters required for indexing in the second round of PCR. Reactions (20 µL) consisted of 1 × KAPA HiFi HotStart ReadyMix (Roche), 500 nM each of forward and reverse primer, and 5 ng template DNA. Thermal cycling conditions were as follows: 95°C for 3 min; followed by 18 cycles of 95°C for 30 s, 50°C for 30 s and 72°C for 30 s; and finally 72°C for 5 min. PCR products were purified using the Agencourt AMPure XP system (1× volume of AMPure beads) and eluted in 50 µL 10 mM Tris-HCl buffer (pH 8.0). The second round of PCRs (30 µL) contained 1 × KAPA HiFi HotStart ReadyMix, 3 µL each of i5 and i7 Nextera XT indexing oligos (Illumina), and 3 µL of purified first-round PCR product. Thermal cycling conditions were as follows: 95°C for 3 min; followed by eight cycles of 95°C for 30 s, 55°C for 30 s and 72°C for 30 s; and finally, 72°C for 5 min. Following purification using 1× AMPure beads, DNA concentrations were measured with the Quant-iT PicoGreen dsDNA Assay Kit and amplicon libraries were pooled at equimolar concentration. The pooled library was supplemented with phiX DNA (30% final concentration) and sequenced on a MiSeq instrument using V2 chemistry (2 × 251 bp reads).
All sequencing data have been deposited in NCBI’s Sequence Read Archive repository under BioProject PRJNA715597.
Other method information
Reagents, measuring instruments and measurement parameters, sequencing data processing and analysis, and lipid data analysis are given in Supplementary Tables S17.