Archives of Toxicology

, Volume 93, Issue 3, pp 603–613 | Cite as

N-(2-Hydroxyethyl)-l-valyl-l-leucine in rat urine as a hydrolytic cleavage product of ethylene oxide adduct with globin

  • Jaroslav MrázEmail author
  • Iveta Hanzlíková
  • Igor Linhart
  • Šárka Dušková
  • Ludmila Dabrowská
  • Kamil Hejl
Toxicokinetics and Metabolism


Ethylene oxide (EO), a genotoxic industrial chemical and sterilant, forms covalent adducts with DNA and also with nucleophilic amino acids in proteins. The adduct with N-terminal valine in globin [N-(2-hydroxyethyl)valine (HEV)] has been used in biomonitoring of cumulative exposures to EO. Here we studied in rats the fate of EO-adducted N-termini of globin after life termination of the erythrocytes. Rat erythrocytes were incubated with EO to produce the HEV levels in globin at 0.4–13.2 µmol/g as determined after acidic hydrolysis. Alternative hydrolysis of the isolated globin with enzyme pronase afforded N-(2-hydroxyethyl)-l-valyl-l-leucine (HEVL) and N-(2-hydroxyethyl)-l-valyl-l-histidine (HEVH), the EO-adducted N-terminal dipeptides of rat globin α- and β-chains, respectively. The ratio of HEVL/HEVH (1:3) reflected higher reactivity of EO with the β-chain. The EO-modified erythrocytes were then given intravenously to the recipient rats. HEVL and HEVH were found to be the ultimate cleavage products excreted in the rat urine. Finally, rats were dosed intraperitoneally with EO, 50 mg/kg. Herein, the initial level of globin-bound HEVL (11.7 ± 1.3 nmol/g) decreased almost linearly over 60 days corresponding to the life span of rat erythrocytes. Daily urinary excretion of HEVL was almost constant for 30–40 days, decreasing faster in the subsequent phase of elimination. Recoveries of the total urinary HEVL from its globin-bound form were 84 ± 6% and 101 ± 17% after administrations of EO and the EO-modified erythrocytes, respectively. In conclusion, urinary HEVL appears to be a promising novel non-invasive biomarker of human exposures to EO.


Ethylene oxide Globin adducts Hydrolytic cleavage Dipeptidic urinary adducts Biomonitoring 



The study was supported by the Grant NT13401-4/2012 from Internal Grant Agency of the Czech Ministry of Health, and by the Czech Ministry of Health, Program DRO (National Institute of Public Health—NIPH, IN 75010330). Purchase of the principal analytical instrument, Orbitrap Q Exactive mass spectrometer, was supported by European Regional Development Fund (ERDF), Integrated Operational Programme Reg. No. CZ.1.06/3.2.01/11.08435. Skilled technical assistance of Hana Chrástecká, Monika Tvrdíková, and Radka Vajtrová (all from NIPH) is gratefully acknowledged.

Compliance with ethical standards

Research involving human and/or animal participants

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. The experiments were approved by the Committee for Animal Protection of the Czech Ministry of Health.

Human data

The manuscript does not contain clinical studies or patient data.

Conflict of interest

The authors declare that they have no conflict of interest.


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Centre of Occupational HealthNational Institute of Public HealthPragueCzech Republic
  2. 2.Department of Organic Chemistry, Faculty of Chemical TechnologyUniversity of Chemistry and TechnologyPragueCzech Republic

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