Differential mobility spectrometry (DMS) reveals the elevation of urinary acetylcarnitine in non‐human primates (NHPs) exposed to radiation

Abstract

Acetylcarnitine has been identified as one of several urinary biomarkers indicative of radiation exposure in adult rhesus macaque monkeys (non-human primates, NHPs). Previous work has demonstrated an up-regulated dose-response profile in a balanced male/female NHP cohort. As a contribution toward the development of metabolomics-based radiation biodosimetry in human populations and other applications of acetylcarnitine screening, we have developed a quantitative, high-throughput method for the analysis of acetylcarnitine. We employed the Sciex SelexIon DMS-MS/MS QTRAP 5500 platform coupled to flow injection analysis (FIA), thereby allowing for fast analysis times of less than 0.5 minutes per injection with no chromatographic separation. Ethyl acetate is used as a DMS modifier to reduce matrix chemical background. We have measured NHP urinary acetylcarnitine from the male cohorts that were exposed to the following radiation levels: control, 2, 4, 6, 7, and 10 Gy. Biological variability, along with calibration accuracy of the FIA-DMS-MS/MS method, indicates LOQ of 20 μM, with observed biological levels on the order of 600 μM and control levels near 10 μM. There is an apparent onset of intensified response in the transition from 6 to 10 Gy. The results demonstrate that FIA-DMS-MS/MS is a rapid, quantitative technique that can be utilized for the analysis of urinary biomarker levels for radiation biodosimetry.

Differential Mobility Spectrometry-Mass Spectrometry (DMS-MS) in Radiation Biodosimetry: Rapid and High-Throughput Quantitation of Multiple Radiation Biomarkers in Nonhuman Primate Urine

“…Our quantitative demonstration of a TML radiation response is consistent with its role as a carnitine precursor since acyl carnitines are also known to be markers of radiation exposure as shown by quantitative measurements in our recent paper which also discusses the matrix effect on calibration [67], and with its observation as a marker in human TBI patients [22]. …”

“…100-fold dynamic range were constructed. Because sufficient NHP control urine was not available and since matrix effect comparisons supporting this substitution will be available in another paper from this group [67], human urine solutions containing various concentrations of each analyte in the range of 10–1000 μM were prepared including isotopically labeled standards (IS). As shown in Figure 2, good linearity with R 2 values of 0.99 or better were obtained for most compounds.…”

Nonhuman Primates with Acute Radiation Syndrome: Results from a Global Serum Metabolomics Study after 7.2 Gy Total-Body Irradiation

“…Similarly, levels of acetylcarnitine (P <.001, 1.96 fold change) and propionylcarnitine (P <.001, 4.56 fold change) were higher at 24 h, as well as 96 h (P = 0.022, 1.65 fold change) for acetylcarnitine and 36 (P <.001, 4.62 fold change) and 96 h (P < 0.036, 1.60 fold change) for propionylcarnitine. Carnitine and its corresponding acyl ester compounds have been identified and quantified in numerous studies and can show drastic dose-dependent changes due to radiation exposure (10,25). These compounds are integral for fatty acid transportation across mitochondrial membranes for subsequent lipid metabolism by enzymatic β oxidation.…”

Temporal Effects on Radiation Responses in Nonhuman Primates: Identification of Biofluid Small Molecule Signatures by Gas Chromatography – Mass Spectrometry Metabolomics

“…While longer run times (~30 min vs. ~5-10 min) and sample preparation for required derivatization (~60 min vs. ~5 min) limits its direct use in biodosimetry for time-sensitive emergency situations, GC-MS serves as a complementary platform to LC-MS providing more depth into metabolite coverage, such as the TCA cycle intermediates described in this study. Subsequent compound analyses can be further refined for high-throughput targeted platforms more suited to biodosimetry, such as differential mobility spectrometry (DMS) MS [63,64] or LC-MS [24]. In this study, we examined global small molecule signatures in NHP biofluids (urine and serum) from 1 -60 d after a 4 Gy γ -ray TBI exposure and determined fold changes in urinary TCA cycle intermediates from 1 -7 d using GC-MS platforms.…”