Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by ¹H NMR Spectroscopy

1. Colonization of germ-free animals and sample collection

1. Remove germ-free animals from isolators and house them in a conventional husbandry room in cages equipped with filter in front of the conventional animals which will serve as controls (Figure 1).
2. Mix half of the litter (3 days old) taken from the control conventional cage with the litter of the germ-free animals. Always keep 1/3 of the dirty conventional litter each time it is necessary to renew it in order to maintain a level of bacteria (keep it at least for 3 days).
3. Collect urine in a 1.5 mL microtube by handling the mouse over the tube and help micturition by gently massage the bowel. Snap-freeze immediately in liquid nitrogen. Store at least at -40°C until NMR analysis. A minimum volume of 20 μL is required for acquisition with a 5 mm NMR probe, but it is recommended to use 30 μL to improve quality of metabolic profiling.
4. Animals should be euthanized without use of any anesthetic in order to avoid confounding NMR resonances due to hepatic metabolism of anesthetic compounds (for example, use cervical dislocation followed by confirmation of death by exsanguination)

2. Recommendation for collection of liver biopsy

1. Do not use any product containing alcohol to avoid contamination. Wash tools using water only or saline solution.
2. Do not perforate gall bladder. In case of bile leak, wash tissue immediately with water or saline solution.
3. Collect liver biopsies (about 15-50 mg) from the left lobe as shown in Figure 2. For reproducible biopsies, collect consistently in the center of the left lobe avoiding the peripheral areas where tissue is thinner.
4. Snap freeze biopsies in liquid nitrogen immediately and store them at -80°C until NMR analysis.

3. ¹H NMR acquisition of urine microvolume

1. Prepare 0.2M sodium phosphate buffer solution in D2O (99.8 %), pH 7.4 containing 1 mM 3-(trimethylsilyl)propionic acid-d₄ (TSP).
2. Mix 30 μL of urine with 30 μL of sodium phosphate buffer.
3. Transfer 50 μL of mixed solution into 1.7 mm NMR capillary tube (Figure 3 (2)) using a 50 μL glass syringe equipped with a metal needle (OD 0.5 mm). Be careful to avoid bubbles.
4. Fit the capillary adaptor (Figure 3 (3)) on top of the capillary containing urine sample and place it into a 2.5 mm NMR microtube for 5 mm NMR probe (Figure 3 (1)). Use this combination of tubes as a regular 5 mm NMR tube for spectral acquisition.
5. Use the extraction rod (Figure 3 (4)) to remove capillary from 2.5 mm NMR tube by screwing the capillary adapter gently to pull it out.

4. ¹H HR MAS NMR of liver tissue biopsy: sample preparation

1. MAS rotor components and tools are described in Figure 4.
2. Insert biopsy (about 15-50 mg) into zirconium rotor (Figure 4 (1)) and fill the rest of the volume with pure D₂O for NMR lock. Be careful to not make any bubbles because this would alter the quality of subsequent shimming and quality of data acquisition.
3. Insert 50 μL Teflon spacer (Figure 4 (2)) using the cylindrical screw (Figure 4 (5)). Unscrew it and calibrate it using the depth gauge on the short side (Figure 4 (8)). At this step, it is important to pay a specific attention to the sample because part of it can leak through the spacer hole. If this is the case, then part of the biopsy is destroyed and sample weight is no longer reliable. It is thus necessary to start again from the beginning the preparation of the sample.
4. Place the thead pin (Figure 4 (3)) and screw it gently with the screwdriver (Figure 4 (6)). Dry out any residual water with a piece of tissue.
5. Place the cap (Figure 4 (4)) at the top of the rotor and insert it in the rotor packer (Figure 4 (6)). Press firmly until the cap is in place. There should not be any space left between the rotor and its cap.
6. Mark half of the bottom of the rotor using a black marker pen to allow optical spin rate detection.
7. Place the rotor inside the NMR spectrometer and start spinning at 5 kHz. Acquire ¹H NMR spectrum using CPMG pulse sequence¹³ according to manufacturer’s guidelines.
8. Use α anomeric glucose resonance at 5.22 ppm (doublet) to calibrate NMR spectra.
9. To unpack the rotor, proceed by removing the cap using the cap remover (Figure 4 (9)). Unscrew thead pin and remove Teflon spacer using the cylindrical screw. Wash thoroughly using water and detergent.

5. Representative Results

Gut microbial activity can be monitored using urinary metabolic profiling. A large number of urinary microbial co-metabolites identifiable by ¹H NMR have been described in the literature^7,14-17. These microbial co-metabolites are particularly useful to monitor the colonization process as they provide a quick and noninvasive way to estimate when the newly established ecosystem is stable. Figure 5A clearly illustrates the appearance of gut microbial co-metabolites over the colonization process. This figure shows a urinary metabolic profile obtained by following the procedure described in Step 2 for an animal colonized 20 days using procedure described in Step 1. This animal did not excrete any indoxyl sulfate and very little amounts of phenylacetylglycine (PAG) and p-cresol sulfate at the germ-free state (day 0-blue). As colonization progresses, these 3 markers of protein metabolism by the gut microbiota increase considerably to reach an equilibrium at day 20 (red). This is particularly easy to monitor for a group of animals as illustrated on Figure 5B using the PAG resonance. This diagram was obtained by integrating the area under the resonances highlighted in gray in Figure 5A (δ 7.40-7.43), corresponding to a specific resonance (triplet) of PAG for a group of 7 animals.

¹H High Resolution Magic Angle Spinning (HR MAS) NMR spectroscopy is a non destructive technique that allows quick and reproducible acquisitions of metabolic profiles of any kind of biopsy¹⁸. In this protocol, we used this powerful technique to obtain a hepatic metabolic profile of 2 mice before (blue) and after (red) colonization (Figure 6). This figure illustrates well the information that can be derived from a MAS NMR-based metabolic profile. Numerous amino acids as well as metabolites derived from energetic metabolism such as glucose, glycogen, lactate, triglycerides, (D)-3-hydroxybutyrate and nicotinurate can be visualized. These profiles also contain information relevant to oxidative stress (i.e. ascorbic acid, glutathione), nucleotide metabolism (i.e. inosine, uridine) and methylamine metabolism (i.e. choline, Trimethylamine-N-oxide). In this example, it is very clear that the germ-free mouse displays almost no glycogen and very low amounts of glucose and triglycerides as was previously published⁷.

Figure 1. Overview of the colonization protocol. Germ-free and conventional animals are housed in cages equipped with filters side by side and their litters are exchanged to allow progressive colonization from the conventional gut microbiota (1). Gut microbial activity is monitored using ¹H NMR-based metabolic profiling (2-3). Hepatic metabolism is assessed by ¹H HR MAS NMR-based metabolic profiling (4-5).

Figure 2. Mouse liver anatomy. The liver is displayed such as the flat side of the organ faces the table. For reproducible biopsies, it is advised to always collect samples from the center of the left lobe as indicated by the dashed rectangle.

Figure 3. 1.7 mm NMR capillary kit to work with microvolumes. Key: 1: 2.5 mm NMR microtube, 2: 1.7 mm NMR capillary tube, 3: Capillary adapter, 4: Extraction rod.

Figure 4. MAS rotor equipment. Key: 1: MAS rotor, 2: 50 μL Teflon spacer, 3: Thead pin, 4: cap, 5: cylindrical screw, 6: screwdriver, 7: rotor packer, 8: depth gauge.

Figure 5. Evolution of urinary metabolic profiles during colonization.

1. Zoom on the aromatic region of the spectra between 6.8-7.8 ppm where microbial co-metabolites can be visualized. ¹H NMR spectra were derived from a single individual at day 0 (blue), 4 (green), 15 (orange) and 20 (red) post-colonization. The grey zone corresponds to the area that was integrated to make the diagram in B. Key: 1-MeHistamine: 1-methylhistamine; Indoxyl-S: Indoxyl sulfate; His: Histidine; p-Cresol-S: p-Cresol sulfate; PAG: Phenylacetylglycine.
2. Average PAG concentration during colonization (n=7). Student’s t-test was used to compare the difference in PAG concentration at various time-points: a: p<0.05 compared to day 0; b: p<0.01 compared to day 10.

Figure 6.Typical 600 MHz ¹H HR MAS NMR spectra of liver biopsies derived from germ-free (blue) and ex-germ-free (red) mice. Bold protons are responsible for the triglyceride resonance. Key: 3-HB: 3-hydroxybutyrate, GSH: reduced glutathione, TGs: Triglycerides, TMAO: Trimethylamine-N-oxide.