1H-NMR for the Evaluation of Biofluids Sample Quality in Metabolomic Research *

Standardized sample handling and processing in metabolomic research is crucial to achieving accurate results.  This article explains how the use of standardized nuclear magnetic resonance (NMR) e.g. based on Bruker Avance IVDr platform, to evaluate different sample handling methods, has proven this fact. Thus, standardized 1H-NMR is a fast and reliable method to monitor subtle changes in biofluids sample.

Metabolomic analysis measures all the small molecules present in a sample, including substrates, intermediary metabolites and the end-products of metabolism. It can thus provide a comprehensive picture of body function and status at the time the sample was taken. It also provides important information regarding disease diagnosis, prognosis, treatment monitoring as well as the body’s response to diet or environmental change.

In metabolomics studies, any biofluids can be used, but blood and urine are most commonly used since they can be obtained most readily. Blood and urine metabolomes reflect the metabolic state of the entire organism being studied, and so are influenced by the status of health, disease, and diet. Metabolic profiling is typically achieved using nuclear magnetic resonance spectroscopy (NMR) or chromatography-based mass spectrometry (LC-MS or GC-MS). Although mass spectrometry offers greater sensitivity, NMR is simpler to perform, has minimal sample preparation and has high reproducibility due to standardisation of the sample preparation, measurement and processing¹.

Since the nature and concentration of metabolites present in a sample will form the basis for conclusions to be drawn, it is essential that the chemical composition analysed accurately reflects the sample at the time it was collected. Differences in the workflow may affect the results obtained thereby raising the possibility of inter-laboratory variation. It is therefore important to know exactly how deviations in sample handling and processing may affect the metabolic profile to ensure highly accurate and reproducible analyses.

Risk of Artefacts in Metabolomic Analysis

Metabolomic profiles can be affected by differences in activity levels, food intake, external conditions, and circadian rhythm². Although it is impossible to totally exclude such natural inter-sample variation, the impact of such factors is typically minimized by sampling at a set time of day after fasting.

More controllable is the potential for introducing unwanted variation during sample collection and analysis. A typical metabolomic workflow comprises several key activities each associated with a myriad of possible variations during both the pre-analytical phase (the process of sample collection, duration, and conditions during transport and storage) and the analytical phase(sample preparation, the precise protocol of analytical technique used).

Standard operating procedures (SOPs) for the pre-analytical handling of blood and urine samples for metabolomic studies were introduced in order to standardize certain variables, such as the time before sample preparation or the method of centrifugation^3,4.

SPIDIA Project (Standardisation of generic Pre-analytical Procedures for In vitro DIAgnostics)

In the quest to ensure standardization of metabolomic analyses, nuclear magnetic resonance (NMR) was used to evaluate the impact of different pre-analytical treatments on the quality of urine and blood samples for metabolomic analysis. The initial findings were used as the basis for the European Committee for Standardization (CEN) technical specifications and further investigations have recently been made (SPIDIA4P – Standardisation of generic Pre-analytical Procedures for In vitro DIAgnostics for Personalised Medicine).

NMR was selected as the analytical method to perform these analyses based on its ability to provide highly reproducible data with an high throughput so the effects of different pre-analytical treatments could be reliably compared⁵.

Fasting blood samples were acquired from the repository of the da Vinci European Biobank⁶. Urine samples obtained from healthy subjects (first urine of the morning under fasting conditions) were stored at 2–8 °C and analyzed no more than 2 hours after collection. Each sample was divided and analyzed using different centrifugation and filtration processing methodologies⁷. ¹H NMR spectra for all samples were acquired using a Bruker Avance IVDr 600 MHz system and following the SOPs for sample preparation and analysis.

Sources of alteration of the urinary metabolomic profiles

In addition to the presence of cells, it is possible that ongoing chemical and enzymatic reactions can alter the content of urine despite only small amounts of enzymes being present. Indeed, of some signals intensity’s as well as the appearance of new metabolites were detected in both processed and unprocessed urine samples⁷. Typically, acetate, succinate, and creatine increased over time, while pyruvate, creatinine, and 2-oxoglutarate decreased.

The main source of change was shown to be redox reactions that, although not completely avoidable, can be minimized by maintaining the samples at low temperatures throughout the analytical and pre-analytical processes and reducing exposure to air. The presence of azide in the NMR buffer did not influence the extent of these changes. Samples stored at −80 °C were found to be well preserved even after 5 years⁷.

Blood Serum and Plasma

Red blood cell activity caused concentrations changes of important metabolites, such as glucose, lactate, and pyruvate, so samples should be processed to remove cells within 30 minutes of collection. As with urine, there were also changes due to oxidation reactions, which affected the concentrations of other metabolites, such as proline and citrate⁷. Similarly, keeping blood samples at low temperatures throughout the analytical and pre-analytical processes reduced changes in the composition. Blood samples stored at −80 °C remained well preserved after 5 years.

In addition, Ficoll gradient centrifugation was shown to strongly interfere with the metabolomic profile of a blood sample, obscuring important metabolites, including glucose and lactate⁷. Fewer spectral signals were lost when EDTA and citrate were used.

Overall, these latest results further confirm the validity of the CEN recommendation document. In addition, the authors confirmed that 1H-NMR is a fast and reliable method for the evaluation of sample quality and validation of sample handling and storage procedures.

References

1. Markley JL, et al. Curr. Biotechnol. 2017;43:34e40
2. Giskeødegård GF, et al. Sci. Rep. 2015;5:14843
3. Bernini P, et al. J. Biomol. NMR 2011;49:231‑243. https://doi.org/10.1007/s10858-011-9489-1
4. Emwas A-H, et al. Metabolomics 2015;11:872–894. https://doi.org/10.1007/s11306-014-0746-7.
5. Takis PG, et al. Trac Trends Anal Chem 2019;120:115300 https://doi.org/10.1016/j.trac.2018.10.036.
6. Ghini V, et al. Metabolomics 2015;11:1769–78. https://doi.org/10.1007/s11306-015-0832-5.
7. Ghini V, et al. New Biotechnology 2019;52:25–34. https://doi.org/10.1016/j.nbt.2019.04.004