In metabolic research, standardized sample handling and processing is vital to attaining accurate results. This article describes how this fact is proven through the use of standardized nuclear magnetic resonance (NMR) for example, based on Bruker Avance IVDr platform, to assess a variety of sample handling techniques. Thus, standardized 1H-NMR is a fast and reliable technique for monitoring subtle variations in biofluids samples.
Metabolomic analysis measures all the small molecules in a sample at that present time, inclusive of substrates, intermediary metabolites and the end-products of metabolism. Thus, it can supply a comprehensive picture of body function and status at the time the sample was taken. It also offers important information with regards to diagnosis of disease, prognosis, treatment monitoring in addition to the body’s reaction to diet or environmental change.
For metabolomics research, any biofluids can be utilized, but blood and urine are most frequently used because they can be found most readily. Blood and urine metabolomes echo the metabolic state of the whole organism being studied, and so are influenced by the status of health, disease, and diet.
Typically, metabolic profiling is accomplished with nuclear magnetic resonance spectroscopy (NMR) or chromatography-based mass spectrometry (LC-MS or GC-MS). Although mass spectrometry provides higher sensitivity, it is simpler to perform NMR, the sample preparation is minimal and it has high reproducibility because of the standardization of the sample preparation, measurement and processing1.
The nature and concentration of metabolites existing in a sample will create the foundations for conclusions to be determined, so it is vital that the chemical composition analysed accurately reflects the sample at the time of collection.
The results obtained may be affected by the variances in the workflow, therefore the possibility of inter-laboratory variation is raised. Due to this, it is essential to know precisely how deviations in sample handling and processing could affect the metabolic profile to guarantee analyses that are extremely accurate and reproducible.
Metabolic analysis: Risk of artefacts
Metabolomic profiles can be influenced by variations in activity levels, food consumption, external surroundings, and circadian rhythm2. It isn’t possible to completely exclude such natural inter-sample disparity, but the influence of such factors is usually minimized by sampling at a specific time of day following fasting.
Increased control gives the possibility of introducing unwelcome variation throughout sample collection and analysis. A characteristic metabolomic workflow consists of many main activities that are each linked with a myriad of potential variations throughout both the pre-analytical phase (the process of sample collection, duration, and conditions during transport and storage) and the analytical phase (preparation of samples, the precise protocol of analytical method used).
Metabolomic studies had standard operating procedures (SOPs) for the pre-analytical handling of blood and urine samples introduced in order to standardize specific variables, like the time prior to sample preparation or the process of centrifugation3,4.
SPIDIA project (Standardization of generic Pre-analytical Procedures for In vitro DIAgnostics)
In the search to guarantee standardization of metabolomic analyses, nuclear magnetic resonance (NMR) was utilized to assess the impact of a variety of pre-analytical treatments on the quality of urine and blood samples for metabolomic analysis.
The preliminary findings were used as the foundation for the European Committee for Standardization (CEN) technical specifications and additional research has been completed recently (SPIDIA4P - Standardization of generic Pre-analytical Procedures for In vitro DIAgnostics for Personalised Medicine).
NMR was chosen as the analytical technique to complete these analyses due to its ability to offer highly reproducible data with an high throughput so reliable comparisons of the effects of different pre-analytical treatments could be made5.
Fasting blood samples were attained from the repository of the da Vinci European Biobank6. Urine samples were attained from healthy subjects (first urine of the morning under fasting conditions) were stored at 2–8 °C and analyzed no more than 2 hours following collection.
Every sample was divided and analyzed with varying centrifugation and filtration processing methods7. NMR spectra for all samples were attained with a Bruker Avance IVDr 600 MHz system and after the SOPs for sample preparation and analysis.
Sources of variation of the urinary metabolomic profiles
As well as the incidence of cells, there is potential that continuing chemical and enzymatic reactions can modify the content of urine in spite of the fact that only small amounts of enzymes are present.
Some signals intensity’s in addition to the presence of new metabolites were distinguished in both processed and unprocessed urine samples7. Characteristically, acetate, succinate, and creatine increased over time, while decreases were seen in pyruvate, creatinine, and 2-oxoglutarate.
The primary source of change was revealed to be redox reactions that, although not entirely preventable, can be minimized by keeping the samples at low temperatures during the analytical and pre-analytical procedures and minimizing exposure to air. The presence of azide in the NMR buffer did not influence the level of these changes. Samples kept at −80 °C were discovered to be well preserved even after a period of 5 years7.
Blood serum and plasma
Red blood cell activity led to concentrations variations of vital metabolites, like glucose, lactate, and pyruvate. Due to this, samples should be processed to eradicate cells within 30 minutes of collection. Similar to urine, there were also changes as a result of oxidation reactions, which influenced the concentrations of other metabolites, like proline and citrate7.
Likewise, storing blood samples at low temperatures during the analytical and pre-analytical procedures lessened changes in the composition. Blood samples kept at −80 °C stayed well preserved after a period of 5 years.
Additionally, Ficoll gradient centrifugation was revealed to strongly affect the metabolomic profile of a blood sample, concealing vital metabolites, inclusive of glucose and lactate7. Less spectral signals were lost when EDTA and citrate were utilized.
In summary, these most recent results further verify the validity of the CEN recommendation document. Additionally, the authors verified that 1H-NMR is a quick and reliable technique for the assessment of sample quality and validation of sample handling and storage procedures.
References
- Markley JL, et al. Curr. Opin. Biotechnol. 2017;43:34e40
- Giskeødegård GF, et al. Sci. Rep. 2015;5:14843
- Bernini P, et al. J. Biomol. NMR 2011;49:231243. https://doi.org/10.1007/s10858-011-9489-1
- Emwas A-H, et al. Metabolomics 2015;11:872–894. https://doi.org/10.1007/s11306-014-0746-7.
- Takis PG, et al. Trac Trends Anal Chem 2019;120:115300 https://doi.org/10.1016/j.trac.2018.10.036.
- Ghini V, et al. Metabolomics 2015;11:1769–78. https://doi.org/10.1007/s11306-015-0832-5.
- Ghini V, et al. New Biotechnology 2019;52:25–34. https://doi.org/10.1016/j.nbt.2019.04.004
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