Pyrrole adducts in globin and plasma of workers exposed to hexane

  • Gaku IchiharaEmail author
  • Venkataraman Amarnath
  • Holly L. Valentine
  • Tatsuya Takeshita
  • Kanehisa Morimoto
  • Tomotaka Sobue
  • Toshio Kawai
  • William M. Valentine
Original Article



Urinary excretion of 2,5-hexanedione is currently used to estimate the exposure levels of hexane occurring to an individual during the previous work shift. However, because hexane exposures and urinary 2,5-hexanedione levels can vary considerably from day to day, and subchronic to chronic exposures to hexane are required to produce neuropathy, this biomarker may not accurately reflect the risk of an individual for developing hexane neuropathy. This investigation examines the potential of hexane-derived pyrrole adducts produced on globin and plasma proteins as markers for integrating cumulative exposures. Because the pyrrole markers incorporate bioactivation of hexane to 2,5-hexandione and the initial step of protein adduction involved in hexane-induced neuropathy, they potentially can serve as biomarkers of effect through reflecting pathogenetic events within the nervous system. Additionally, pyrrole formation is an irreversible reaction suggesting that hexane-derived protein pyrroles can be used to assess cumulative exposures to provide a better characterization of individual susceptibilities.


To examine the utility of the proposed markers, blood samples were obtained from eleven workers who used hexane for granulating metal powders in a slurry to produce metal machining die tools and four non-exposed volunteers. Globin and plasma were isolated, and the proteins were digested using pepsin, reacted with Ehrlich’s reagent and the level of pyrrole adducts were determined by absorbance at 530 nm. To determine the dose–response curve and dynamic range of the assay, erythrocytes were incubated with a range of 2,5-hexanedione concentrations and the net absorbance at 530 nm of isolated globin was measured.


Pyrrole was detected in both the globin and plasma samples of the workers exposed to hexane and the levels of pyrroles in plasma were positively correlated with the levels of pyrroles in globin for most of the workers.


This investigation demonstrates that detectable levels of hexane-derived protein pyrrole adducts are produced on peripheral proteins following occupational exposures to hexane and supports the utility of measuring pyrroles for integrating cumulative exposures to hexane.


Hexane Pyrrole Neurotoxicity Biomarker Adduct 



Our sincere thanks to Dr. Doyle G. Graham (Vanderbilt University Medical Center, TN, USA), Dr. Yasutaka Ogawa, Dr. Naomi Hisanaga (National Institute of Industrial Health, Kanagawa, Japan) and Dr. Yasuhiro Takeuchi (Emeritus Professor of Nagoya University, Nagoya, Japan).

Author contributions

GI and WMV deigned the project and wrote the manuscript. GI, VA and HLV measured pyrrole adducts and protein. TT, KM and TS conducted the field-work. TK measured urinary 2,5-hexanedione. VA drew the figure of pyrrole formation and detection.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Informed consent

All assays were performed with the subjects’ informed consent following the Helsinki Declaration. The study protocol was approved by the ethical committee of Tokyo University of Science.


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Copyright information

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

Authors and Affiliations

  • Gaku Ichihara
    • 1
    Email author
  • Venkataraman Amarnath
    • 2
  • Holly L. Valentine
    • 2
  • Tatsuya Takeshita
    • 3
  • Kanehisa Morimoto
    • 4
  • Tomotaka Sobue
    • 4
  • Toshio Kawai
    • 5
  • William M. Valentine
    • 2
  1. 1.Department of Occupational and Environmental Health, Faculty of Pharmaceutical SciencesTokyo University of ScienceNodaJapan
  2. 2.Department of Pathology, Microbiology and ImmunologyVanderbilt University Medical CenterNashvilleUSA
  3. 3.Department of Public HealthWakayama Medical UniversityWakayamaJapan
  4. 4.Department of Social and Environmental MedicineOsaka University Graduate School of MedicineOsakaJapan
  5. 5.Osaka Occupational Health Service CenterOsakaJapan

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