Identification and Quantification of Deranged Metabolites in Critically Ill Patients Using NMR-Based Metabolomics

Our research uses NMR-based metabolomics to identify dysregulated metabolites and biochemical pathways which get altered in critically ill ICU patients, and we have also included patients of ARDS, which is a severe lung condition. By profiling and comparing the metabolic signatures, we aim to uncover the biochemical disturbances which are linked to disease progression and customize treatments to improve outcomes. Actually, we are trying to address the gap in understanding metabolic dysregulation in all categories of ARDS patients, which includes mild, moderate, and severe ARDS patients.

To improve our insights, for a better understanding of the disease, its progression, with the overall aim of improving the patient outcome by providing precision and personalized medicine. NMR offers easy sample preparation, preserving serum integrity for reanalysis and detecting known and unknown metabolites without chemical derivatization. It provides reproducible, quantitative data, ensuring reliable metabolite measuring.

Unlike targeted approaches, NMR enables sensitive, unbiased exploration of metabolic changes, making it ideal for discovering signatures and pathways in disease progression. Our finding will enhance ARDS research by uncovering metabolic changes in different stages, which are understudied. Using NMR-based metabolomics, we aim to identify dysregulated metabolites and pathways, leading to potential signatures for early diagnosis, prognosis, more timely prediction and treatments.

This approach advances personalized management and deepens knowledge in multi-state metabolic profiling. Our results paved the way for new scientific questions, such as:How do specific metabolites identified in different stages of ARDS influence disease progressions? Can these dysregulated pathways be targeted for early therapeutic interventions?

Additionally, how do these metabolic changes vary across other respiratory or inflammatory conditions? To begin, using a sterile needle, collect two milliliters of blood from the arteries of a patient diagnosed with mild or moderate acute respiratory distress syndrome or ARDS. Transfer blood into a sterile vial and allow it to clot for 30 minutes at room temperature.

Centrifuge the clotted blood at 3, 100 G for 15 minutes to separate the serum. Transfer the serum into sterile microcentrifuge tubes, making aliquots of 400 to 500 microliters. Label the tubes with the patient's details and store them at 80 degrees Celsius.

For NMR experiments, mix 250 microliters of the serum aliquot with 250 microliters of saline phosphate buffer to minimize pH variation. Vortex the mixture for approximately 15 seconds to ensure homogeneous mixing. Transfer the prepared sample into a clean NMR tube with a coaxial insert containing TSP at a concentration of 0.05 millimolar.

Next, place the NMR tube into the magnet. Type WRPA and select the appropriate experiment number to set up a proton experiment with the CPMG pulse program. Click on the Title option to enter the patient's name and other necessary details.

Type lock and press Enter to lock the magnetic field. Then, select the options 90%water and 10%deuterium oxide. Then, type ATMM and press Enter to perform tuning and matching of the probe.

Next, from the Acquisition Parameters option, select the parameters as 64, 000 points over 128 scans to collect optimization data. Set the relaxation delay of five seconds, a spectral sweep width of 12 PPM, and an echo time of 200 microseconds, repeated 300 times. Apply line broadening of 0.3 hertz using an exponential window function.

Next, using the NMR processing and analysis tools, acquire the NMR spectra. Once the final spectra are obtained, type APK and ABSN to perform phase correction and baseline correction respectively. Then, click on the Calibrate Axis option in the upper panel and calibrate the TSP peak to 0 PPM, either automatically or manually.

Finally, transfer the acquired data from the NMR system to the workstation for further analysis. After acquiring NMR spectra of patients diagnosed with mild or moderate ARDS, open NMR metabolite quantification software for data processing. To perform manual baseline correction using the Whitaker method, open the spectral file in the processor module of NMR metabolite quantification software.

Select baseline correction, then choose the Whitaker method. Place dots at the base of peaks in the entire spectra. Once completed, save the baseline-corrected spectral files in a single folder.

Next, to perform spectral binning using the Profiler module, select the Tools, then Batch Process, followed by Spectral Binning. To generate the final binning sheet for statistical analysis, select the folder with all baseline-corrected spectral files. Divide the spectra into equal-sized bins with a defined spectral width ranging from 0.01 to 0.04 parts per million.

Specify the bucket size and the start and end PPM values. Choose a folder to save the output binning sheets. Exclude the chemical shift regions corresponding to water and the solvent TSP to prevent spectral interference.

The final spreadsheet will display sample names in one row and bin values with corresponding PPM in other rows. Before performing statistical analysis, normalize the data using some normalization, log transformation, and Pareto scaling. For multivariate analysis, perform Principal Component Analysis, Partial Least Squares Discriminant Analysis, and Orthogonal Partial Least Squares Discriminant Analysis to observe discrimination between the groups, yielding variable importance of projection scores for every metabolite responsible for the difference in the metabolic profile.

Perform Student's t-test to identify significant metabolites with a p-value of less than 0.05. T-tests produce box whisker plots displaying differences in metabolite concentrations. Then, conduct biomarker analysis to generate an area under the receiver operating characteristic curve graph.

Select the final list of significant metabolites based on a variable importance of projection score greater than one, a Bonferroni-corrected p-value less than 0.05, and an area under the curve greater than 0.8. Using the Human Metabolome Database, identify the metabolites corresponding to specific PPM values. Before beginning the quantification, using the processor module, calibrate the reference compound selected for the study.

Click on the Calibrate Chemical Shift Indicator option and enter the concentration of the reference compound. Drag the software peak onto the spectral reference peak and adjust its height and width. Once the spectral reference peaks align with the software's peak, click Accept.

Next, open the desired spectra in the Profiler module and select the appropriate compound library for study. At the bottom of the screen, a list of metabolites will be displayed. Select the metabolite of interest from the list.

Click on the PPM value at the top corner of the screen. Zoom in on the spectra to the specific PPM scale of the metabolite. At this point, two peaks appear on the screen, one representing the recorded data and the other, shown as a dotted line, representing the software peak.

Drag the software peak to align it with the recorded peak, adjusting the height and position to match. Once the peaks are properly aligned, read the concentration of the metabolite from the value displayed under the heading Concentration in millimolar. To generate the output file, under the Tools menu, click on the Batch Operations option and specify the desired folder for saving the output file.

Principal Component Analysis displayed distinct clustering between mild and moderate ARDS patient groups. Orthogonal Partial Least Squares Discriminant Analysis revealed a clear separation between the mild and moderate ARDS groups, emphasizing metabolic differences. Student's t-test and biomarker analysis revealed key dysregulated metabolites between mild and moderate ARDS.

Lactate and isoleucine were identified as significantly dysregulated metabolites correlating with ARDS severity.