2-Methyl-pentanoyl-carnitine (2-MPC): a urine biomarker for patent Ascaris lumbricoides infection

Infections with intestinal worms, such as Ascaris lumbricoides, affect hundreds of millions of people in all tropical and subtropical regions of the world. Through large-scale deworming programs, World Health Organization aims to reduce moderate-to-heavy intensity infections below 1%. Current diagnosis and monitoring of these control programs are solely based on the detection of worm eggs in stool. Here we describe how metabolome analysis was used to identify the A. lumbricoides-specific urine biomarker 2-methyl pentanoyl carnitine (2-MPC). This biomarker was found to be 85.7% accurate in determining infection and 90.5% accurate in determining a moderate-to-heavy infection. Our results also demonstrate that there is a correlation between 2-MPC levels in urine and A. lumbricoides DNA detected in stool. Furthermore, the levels of 2-MPC in urine were shown to rapidly and strongly decrease upon administration of a standard treatment (single oral dose of 400 mg albendazole). In an Ascaris suum infection model in pigs, it was found that, although 2-MPC levels were much lower compared to humans, there was a significant association between urinary 2-MPC levels and both worm counts (p = 0.023) and the number of eggs per gram (epg) counts (p < 0.001). This report demonstrates that urinary 2-MPC can be considered an A. lumbricoides-specific biomarker that can be used to monitor infection intensity.

S. mansoni pos (n) 11 6 6 9 Suppl. Figures   Fig. S1. Co-injection experiment hexanoyl carnitine  S4. Correlation between A. lumbricoides FECs (expressed in epg) and A. lumbricoides DNA detection in stool collected in Kenya (expressed in A. lumbricoides copies/reaction) was used to determine a cut-off of 700 copies/reaction that could be used to identify subjects with moderate-to-high infection.

Preparation of QC samples for metabolomics
A quality control (QC) pool was constructed by collecting 50 or 100 μL of all the plasma or urine samples, respectively, that were used for the untargeted discovery approaches.
Subsequently, this QC pool was divided into aliquots to acquire representative QC samples.
QC samples were prepared simultaneously along with study samples and were analyzed throughout the LC-MS analysis sequences every five study samples. Since these samples do not contain any biological variability, they can be considered as technical replicates. For both plasma and urine, study and QC samples were prepared in random order. Blank extracts were prepared simultaneously along with study samples and were analyzed before the LC-MS analysis sequences to check the overall contamination in the analytical pipeline.

Sample preparation for RP-LC Q-TOF-MS based metabolomics
The frozen urine samples were thawed on ice and centrifuged at 20,627 x g for 10 min. Samples (v/v) and transferred to a new Eppendorf tube. The samples were centrifuged for 10 min at 20,627 x g, after which 80 μL of supernatant was transferred to a vial with glass insert for LC-MS analysis.

RP-LC Q-TOF-MS based metabolomics
The LC-MS method was adapted from Boelaert et al. 1  High-resolution accurate mass spectra and fragmentation spectra were obtained with an Agilent Statistically significant features (p-value < 0.05) containing the appropriate fold change differences were exported and once again extracted from the raw data, a process named recursion. Here, all the extracted ion chromatograms were manually checked, and false positives were excluded. All compounds checked in the recursion step were again subjected to the Mann-Whitney-U test. In these analyses correction for multiple testing was performed with the Benjamini Hochberg false discovery rate.

Quality of analysis of the metabolomics approach
The validity of the performed analyses was monitored in both a targeted and a non-targeted manner using the QC samples. Targeted monitoring was performed by determining the error of the measurement on signal intensity (peak area), retention time and mass accuracy for a list of 18-22 randomly selected metabolites. Peak area fluctuations, originating from both the sample preparation step and the LC-MS analysis, are typically below 15% relative standard deviation 2 . Chromatographic retention time reproducibility is in general satisfactory and less than 1 RSD%. Also, high mass accuracy (< 5ppm) was obtained for all analyses.
Apart from this targeted approach, the reproducibility of the applied metabolomics analysis was examined in a more comprehensive way by calculating the error on all detected features in the QC samples and representing the acquired RSD distribution as depicted in S3 Fig. For all analyses performed, > 75% of all features had an RSD below 30%, which can be defined as the upper limit for untargeted or discovery metabolomics analysis 3 .

Metabolite identification
Metabolite identification results from an identification strategy that fully exploits the features of the Q-TOF MS system. Generation of molecular formulas, based on accurate mass, isotope abundance, and isotope spacing both in positive and negative ionization mode, was

Infrared ion spectroscopy (IRIS) analysis
Details of the hardware modifications to the quadrupole ion trap mass spectrometer and the synchronization with the IR laser are described elsewhere 5  ). The yield was plotted against the IR frequency to generate an IR spectrum.
The IR frequency was calibrated using a grating spectrometer, and the IR yield was linearly corrected for frequency-dependent variations in the laser pulse energy.

a) 2-Methyl-pentanoyl-L-carnitine synthesis
L-Carnitine was dissolved in TFA (4 mL). The solution was added into 2-methyl pentanoyl chloride. The reaction mixture was heated at 50 o C for 12 hrs, and then was evaporated to dryness in vacuo to give a crude product containing TFA as a thick oil.
Part of the of the crude product (thick gum, 888 mg containing TFA) was saved. Remaining crude product (~400 mg) was dissolved in water (5 ml) to give an acidic solution and then was treated with basic resin (Amberlyst® A21 free base resin) until pH became neutral (pH 6-7).
The solution was then filtrated and evaporated to give a thick colorless gum, and then foam (334 mg).