We support all aspects of metabolic phenotyping, from sample to data
The metabolic phenotyping of human biological samples provides a metabolic picture which is reflective of the status of the patient at the time that the sample was collected. This information can then be used to improve our understanding of a disease and facilitate the discovery of mechanistic, prognostic and diagnostic biomarkers.
The CPC provides a collaborative and comprehensive service to handle all aspects of metabonomic research, including sample preparation, sample analysis and data pre-processing. Additionally, we are able to offer statistical analysis, data fusion and interpretation.
To obtain meaningful results, researchers need to define the study's objective clearly and have a robust study design. If required, the CPC can help designing the study and selecting the most appropriate analytical platform to answer the research question.
Please consider the following before starting your project
It is critical to define the purpose of the study clearly as it will determine the design and metabonomic approach for the study. For example, if you intend to have a better understanding of a disease and discover biomarkers of the disease progression, global profiling analysis will provide relevant information on affected metabolic pathways and can identify upregulated metabolites on each stage of the disease. If on the other hand, you already know the metabolic pathways affected by a disease, a targeted approach will provide the required in-depth knowledge of specific metabolites or metabolite classes.
It is necessary to have a robust study design to obtain meaningful metabolic profiles. An appropriate patient population must be selected to address the biological question and to get balanced and homogeneous groups. Each of the biological hypothesis tested, should have an adequate control group and enough samples collected to obtain statistically significant results. The biological question will also determine the analytical approach (global vs. targeted) and whether any method development will be required. It is imperative to collect a sample matrix or matrices relevant to the biological question and at a sufficient volume for the subsequent analyses. Sample collection and handling has to be consistent and degradation should be minimised throughout the investigation. Finally, it is also important to reduce the presence of confounding factors including uncontrolled dietary intake, unrecorded xenobiotic administration, ethanol contamination, inconsistent application of the protocol, etc. Too many uncontrolled confounding factors can skew the results.
- Appropriate control group?
- Adequate number of patients?
- Homogenous groups?
- Food/drinks intake
- Cleaning reagents
- Sample type?
- Consistent time-points?
- Consistent sample collection and handling?
- Sufficient volume?
- Global profiling or targeted assay?
- Method development required?
Importance of using the correct protocol and approach
The high sensitivity of metabonomic investigations requires adherence to strict collection and handling protocols which ensure that there is minimal sample degradation and the maximum amount of information will be retrieved from each metabolic profile. Some general recommendations on sample collection and handling are summarised in the samples section.
Different analytical techniques and approaches can be employed for metabonomic analyses depending on the biological question that needs to be answered. Proton nuclear magnetic resonance (1H NMR) spectroscopy is commonly used as a first stage metabolic screening tool. To increase metabolome coverage, complementary global profiling and targeted assays are followed up using more sensitive mass spectrometry (MS)-based methods.
Main analytical techniques employed in metabolic phenotyping
High-resolution NMR spectroscopy and MS are the techniques applied most frequently in metabolic profiling. Both of them offer quantitative and structural information of a wide range of structurally diverse metabolites simultaneously, providing a metabolic ‘snapshot’ at a particular time point. Both platforms have different analytical strengths and weaknesses and provide complementary information of the sample under study.
NMR spectroscopy, and we mean mainly 1H NMR, provides a universal detector. It is a cost-effective, quantitative, non-destructive, non-invasive and non-equilibrium perturbing technique. It provides detailed information on solution-state molecular structures, based on atom centred nuclear interactions and properties. Other advantages of NMR spectroscopy include minimal sample preparation and fast data acquisition per sample, so high-throughput and automated analysis is possible. Moreover, it is robust, reliable and highly reproducible with slight variation coefficients. The largest disadvantage of NMR spectroscopy is its poor sensitivity in comparison to MS techniques, which limits the number of observable molecular species. However, sometimes changes in low concentration metabolites may lead to indirect changes in higher concentrations metabolites (visible to NMR spectroscopy). In addition to limitations associated with detection limits it also, may suffer from co-resonances.
MS-based metabonomics offers quantitative analyses with high selectivity and sensitivity. The largest disadvantage of MS in comparison with NMR spectroscopy is its lower reproducibility. A mass spectrometer forms charged species, separates those ions according to their mass-to-charge ratio (m/z) and detects them. The combination with a separation technique such as liquid chromatography (LC) reduces the complexity of the mass spectra due to metabolite separation in a time dimension and delivers additional information on the physicochemical properties of the metabolites. For LC-MS analysis, the ionisation technique of choice is electrospray (ESI). Ionisation must be performed in both positive and negative ion modes to obtain a broad coverage of the metabolome. MS-based techniques might require a sample preparation step, and depending on the introduction system and the ionisation technique, specific metabolite classes may be discriminated or metabolites differentially ionised.
Global metabolic profiling vs. targeted approach
For novel clinical studies, a comprehensive analysis of the total metabolite content, also known as metabolome, is key and therefore a discovery strategy with a wide coverage is recommended. This approach is known as global metabolic profiling and ensures that the maximum number of metabolites is analysed in order to obtain an understanding of the general metabolic perturbation associated with a specific biological state or disease. Metabolic profiling can give crucial insights of the disease basis identifying associated biomarkers, and can also help understanding why a treatment is working.
A targeted approach is recommended when the biological question is focused on a specific class of molecules or metabolic pathway (such as the amino acids, bile acids or the transsulfuration pathway). Targeted assays offer in-depth insights of particular biomarkers. Such approaches might require specific sample handling or optimisation of instruments.
As with all studies, it is essential to power the study but it is particularly pertinent in systems metabolism where hundreds or thousands of metabolic endpoints may be studied simultaneously. Therefore for longitudinal or randomised clinical studies, there should be a clear evidence of a power calculation, and around 50 patients is the minimum required to generate a meaningful analysis. For single time point studies or those with limited biofluids or samples, then much larger sample numbers are likely to be required to generate meaningful insights.