Elimination of 15N-thymidine after oral administration in human infants.

BACKGROUND: We have developed a new clinical research approach for the quantification of cellular proliferation in human infants to address unanswered questions about tissue renewal and regeneration. The approach consists of oral 15N-thymidine administration to label cells in S-phase, followed by Multi-isotope Imaging Mass Spectrometry for detection of the incorporated label in cell nuclei. To establish the approach, we performed an observational study to examine uptake and elimination of 15N-thymidine. We compared at-home label administration with in-hospital administration in infants with tetralogy of Fallot, a form of congenital heart disease, and infants with heart failure. METHODS: We examined urine samples from 18 infants who received 15N-thymidine (50 mg/kg body weight) by mouth for five consecutive days. We used Isotope Ratio Mass Spectrometry to determine enrichment of 15N relative to 14N (%) in urine. RESULTS/FINDINGS: 15N-thymidine dose administration produced periodic rises of 15N enrichment in urine. Infants with tetralogy of Fallot had a 3.2-fold increase and infants with heart failure had a 4.3-fold increase in mean peak 15N enrichment over baseline. The mean 15N enrichment was not statistically different between the two patient populations (p = 0.103). The time to peak 15N enrichment in tetralogy of Fallot infants was 6.3 ± 1 hr and in infants with heart failure 7.5 ± 2 hr (mean ± SEM). The duration of significant 15N enrichment after a dose was 18.5 ± 1.7 hr in tetralogy of Fallot and in heart failure 18.2 ± 1.8 hr (mean ± SEM). The time to peak enrichment and duration of enrichment were also not statistically different (p = 0.617 and p = 0.887). CONCLUSIONS: The presented results support two conclusions of significance for future applications: (1) Demonstration that 15N-thymidine label administration at home is equivalent to in-hospital administration. (2) Two different types of heart disease show no differences in 15N-thymidine absorption and elimination. This enables the comparative analysis of cellular proliferation between different types of heart disease.

Simultaneous observation of 2H and 13C enrichment of methyl phosphonic acid via Orbitrap-IRMS with applications to nerve agent forensics.

Quantification of the stable isotopes within a compound aids forensic investigations as it provides a fingerprint which can determine that compound's source substrates, synthetic route, and possible mechanisms of degradation. Previous stable isotope studies have explored 13C and 2H measurements of the sarin precursors methylphosphonic dichloride (DC) and methylphosphonic difluoride (DF) as forensic signatures. However, these measurements required different sample preparations and measurement techniques. Orbitrap isotope ratio mass spectrometry (Orbitrap-IRMS) is a developing technique which can characterize multiple stable isotopes simultaneously. Here, we apply Orbitrap-IRMS to simultaneously observe the 13C and 2H content of methylphosphonic acid (MPA), the hydrolysis product of DC and DF, which can be used as a proxy for the isotopic content of DC and DF. Our method requires 20 min analyses and consumes ≈60 nmol of sample, with precisions of ≈0.9 ‰ (13C) and ≈3.6 ‰ (2H). We apply our method to both commercially acquired MPA and MPA obtained from the hydrolysis of commercially acquired DC. We validate our methods via comparison to elemental-analyzer isotope ratio mass spectrometry (EA-IRMS). The combined 13C and 2H measurement creates a more robust forensic tool than either isotope individually. Our results demonstrate the viability of Orbitrap-IRMS for chemical forensic measurements.