Animal Tissue Sample Collection and Processing in Metabolomic

Compared to body fluids, compounds such as tissue secreted hormones, enzymes and transmitters may be metabolized, excreted or diluted, and the resulting metabolite differences are more easily identified in the corresponding tissues.

To ensure reproducibility of data and comparison of metabolomics results between laboratories, standard operating procedures should be followed at each step of the workflow, especially with regard to sample preparation. Tissue sample preparation strategies need to be optimized and standardized, and the following are recommendations for the preparation of tissue samples from animals metabolomics based on mass spectrometry (MS) techniques.

Extraction Procedure of Animal Tissue Samples

1) Cryogenic treatment

Tissue samples are different from body fluid samples and generally need to be washed immediately after sampling to eliminate blood contamination and then cryoprocessed to achieve metabolic termination. For example, skeletal muscle needs to be washed in physiological saline to remove blood and blotted dry with filter paper, wrapped in aluminum foil in a sealed bag, and quickly placed in a -80°C refrigerator or liquid nitrogen to be stored for assay.

2) Homogenization

To further improve metabolite coverage and reproducibility, samples can be homogenized according to experimental requirements. Mechanical methods such as manual grinding, motorized homogenization and ultrasonic fragmentation are commonly used to fragment tissues for animal tissue-based metabolomics sample processing methods. Some tissues are subject to enzymatic treatment, such as lung and heart tissues.

3) Extraction

Liquid-liquid extraction (LLE): A method that uses different solubility or partition coefficients of solvents to separate mixed metabolites.

Diagrammatic illustration of liquid-liquid extraction (Targuma et al., 2021)

Water-soluble and insoluble metabolites must be extracted in different solvents, and efforts should be made to reduce the amount of contaminants in the vessel. Different extraction methods are selected according to the polarity of metabolites to extract the maximum amount of polar metabolites or non-polar metabolites from the tissues. For example, strongly polar metabolites in muscle tissue are extracted using methanol/water and acetonitrile/water; non-polar metabolites are extracted using chloroform. In general, both hydrophilic and lipophilic extraction solvents are chosen to extract as many metabolites as possible at the same time, such as methanol/chloroform/water and chloroform/methanol. The proportion of chloroform is no more than 20% to avoid phase separation, and it can be used as a carrier to make phospholipids soluble in methanol phase.

Solid phase extraction (SPE): The separation is achieved by exploiting the difference in the interaction of different substances in the solid and liquid phases.

Stages of solid phase extraction (SPE) (Al-Karawi et al., 2016).

Animal tissue samples are adsorbed onto the stationary phase and then eluted in steps using solutions with different elution capacities to achieve separation, purification and enrichment. In general, SPE is commonly used after LLE to remove interfering substances from the extract or to specifically enrich for a class or classes of compounds for targeted metabolomic analysis.

In vivo sampling of animals for sample preparation is more in line with metabolomics and animal welfare concepts, especially in the study of endangered animals. Microdialysis and solid-phase microextraction (SPME) have emerged. Samples taken ex vivo are subject to a chain reaction of oxidation, PH changes, and other activating biological processes that can eventually interfere with metabolite profiles. The microdialysis technique is a combination of perfusion sampling and dialysis techniques and has been gradually refined. With the help of a stereotaxic instrument, microdialysis probes are precisely implanted at a specific location to extract small molecules of metabolites directly from living tissues to achieve dynamic microbiological sampling.

Sometimes an additional evaporation and re-solubilization step is required for undetectable metabolites. Derivatization may also be required for metabolites with high polarity and low volatility within animal tissue samples, such as amino acids, fatty acids, amines, sugars and nucleotides.

Creative Proteomics can provide a one-stop shop for metabolomic analysis of tissue samples. You can send your samples to us and we will perform sample pre-processing prior to analysis. You only need to perform the following operations:

1) Animal tissue samples: If the whole tissue is taken by perfusion method, blood residues in the tissue are removed with pre-cooled deionized water. If partial tissue is taken, rinse off blood residue with pre-chilled deionized water after crushing the tissue.

Human tissue samples: small biopsy samples are quickly rinsed with pre-cooled deionized water to remove blood residues.

2) 250 mg/sample loaded into centrifuge tubes by taking specific sites according to the specific experimental design.

3) Marked and quickly placed in liquid nitrogen for freezing for at least 15 min.

4) Freeze and store at -80°C. Send a sufficient amount of dry ice.

For samples like zebrafish brain tissue, mouse hippocampal tissue and other individual experimental animal tissues with small weight, please consult the technical staff.

References

1. Targuma, Sarem, Patrick B. Njobeh, and Patrick G. Ndungu. "Current applications of magnetic nanomaterials for extraction of mycotoxins, pesticides, and pharmaceuticals in food commodities." Molecules 26.14 (2021): 4284.
2. Al-Karawi, Dheyaa Hussein. "The Investigation of The Electrical Control of Hemimicelles and Admicelles on Gold for Analyte Preconcentration." (2016).