A simplified method for detection of N‐terminal valine adducts in patients receiving treosulfan

Abstract

RATIONALE Treosulfan is a substance that is being studied as part of the conditioning regimen given prior to allogeneic stem cell transplantation in patients with hematological malignancies. It is known to decompose into 1,2:3,4‐diepoxybutane (DEB) under physiologic conditions. In this study, we investigate whether N‐terminal valine adducts can be utilized to monitor differences in DEB formation of patients receiving treosulfan as part of the conditioning regimen for transplantation. METHODS Blood samples were collected from a group of 14 transplant recipients and analyzed for N,N‐(2,3‐dihydroxy‐1,4‐butadiyl)valine (pyr‐Val) and 2,3,4‐trihydroxybutylvaline (THB‐Val) adducts as biomarkers for drug uptake and metabolism before treosulfan treatment and 6 days after treatment. RESULTS A new direct injection liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed and validated prior to clinical analysis. The assay precision was determined by 3 replicate analyses on 3 individual days using control globin spiked with known amounts of pyr‐Val and THB‐Val. The intra‐ and inter‐day precision coefficients of variance (CVs) and accuracy were < 10% and 15%, respectively. In clinical specimens, the means ± SD of pyr‐Val and THB‐Val background were 0.29 ± 0.10 pmol/g HB and 5.17 ± 1.7 pmol/g HB, respectively. CONCLUSIONS These values are similar to those found previously. Treosulfan treatment leads to a significant increase in pyr‐Val and THB‐Val adducts in each patient (Student's t‐test p <0.0001). The mean ± SD amounts of adduct formed were 245.3 ± 89.6 and 210 ± 78.5 pmol/g globin for pyr‐Val and THB‐Val, respectively. Importantly, these results show that this direct injection method can quantitate both background and treosulfan‐induced pyr‐Val and THB‐Val N‐terminal valine globin adducts in humans.

Quo vadis blood protein adductomics?

“…Such imidazoline adducts have been determined for example also with acetaldehyde (Birt et al 1998). ( 4) N-Terminal valine adducts of treosulfan analyzed after trypsin digestion (Boysen et al 2019). The same adduct was formed with diepoxybutane (Kautiainen et al 2000).…”

“…In contrast, the hemoglobin adduct levels found in rats of bicyclic and bifunctional arylamines such as 4,4'-methylenedianiline, 4,4'-methylenebis(2-chloroaniline), 4,4'-oxydianiline, 4,4'-thiodianiline, 3,3'-dichlorobenzidine and benzidine correlate with the carcinogenic potency (Sabbioni and Schutze 1998). Roughly, the mutagenic and carcinogenic potency of arylamines is associated s (Xu et al 2014) HETE-Val, CAS:190187-17-8 t (Boysen et al 2019) CAS:2416700-26-8, main product u (Wheelock et al 2018) v (Schutze et al 1995) w (Padros and to the relative stability of the nitrenium ion, but not necessarily to the hydrolyzable hemoglobin (sulfinamide) adduct levels (Sabbioni and Sepai 1995;Sabbioni and Wild 1992) (Fig. 1S).…”

“… APCI=atmospheric pressure chemical ionization, FITC = fluorescein isothiocyanate, FLD=fluorescence detection, HBFA = heptafluorobutyric anhydride, HRMS=high resolution mass spectrometry, hydr = hydrolysis, NCI=negative chemical ionization, PFPITC = pentafluorophenyl isothiocyanate, PITC = phenylisothiocyanate a Hb b globin c (Gries and Leng 2013 ) CAS:264285-90-7 d (Sachse et al 2017 ) CAS:1531625-56-5 e (Hielscher et al 2017 ) CAS:133278-70-3; e1) (Müller 2013 ) f (von Stedingk et al 2010 ) CAS:252663-74-4; f1) (Yang et al 2018 ); f2) (Schettgen et al 2016 ) g (Yang et al 2018 ) CAS:21768-51-4 h CAS:51078-53-6 i CAS:51078-49-0 j (Carlsson et al 2015 ), only the FITC derivative has a CAS number k (Lewalter et al 2012 ) CAS:15363-84-5 l (Bader et al 2013 ) CAS:223443-77-4 m (von Stedingk et al 2014 ), N -[2-(2-oxo-3-oxazolidinyl)ethyl]-L-valine CAS:173962-82-8 n (CDC-NHANES 2020 ) N-terminal peptide (VHLTPEEK) of the ß-Hb-chain: N-(Hydroxymethyl)-VHLTPEEK (CAS:1307263-38-2), also the terminal amino acids of the α-Hb-chain was reported but not quantified in the CDC-NHANES studies, N-(hydroxymethyl)-VLSPADK, CAS:1307263-39-3; n1) (Yang et al 2017 ) o (Charneira et al 2012 ) CAS:1350434-49-9 p (Käfferlein et al 2016 ) CAS:74310-99-9 q CAS:848640-59-5 r (Mráz et al 2006 ) CAS:84860-36-6 s (Xu et al 2014 ) HETE-Val, CAS:190187-17-8 t (Boysen et al 2019 ) CAS:2416700-26-8, main product u (Wheelock et al 2018 ) v (Schutze et al 1995 ) w (Padros and Pelletier 2001 ); * were validated in the German Working Group “Analyses in Biological Materials of the permanent Senate Commission for the Investigation of Health Hazards of Chemical Compounds in the Work” and the standard operation values are available online ( https://onlinelibrary.wiley.com/doi/book/10.1002/3527600418 ) ** the compounds are commercially available, SciFinder search 23.7.21 …”

“…Applying a similar method, the adduct of treosulfan was used to detect the N-terminal adduct 2,3,4‐trihydroxybutyl-VLSPADK of the reactive intermediate diepoxybutane. After enzymatic digestion, the 7-mer adducted peptide was analyzed by LC–MS/MS (Boysen et al 2019 ). The same approach was used to analyze N-terminal N-acylated and deaminated Val.…”

Effects of GSTT1 Genotype on the Detoxification of 1,3-Butadiene Derived Diepoxide and Formation of Promutagenic DNA–DNA Cross-Links in Human Hapmap Cell Lines

“…Metabolic activation of BD involves oxidation by cytochrome P450s, mainly CYP2E1 and CYP2A6, to several epoxide metabolites, 3,4-epoxy-1-butene (EB), 3,4-epoxy-1,2-butanediol (EBD), and 1,2,3,4-diepoxybutane (DEB) (Scheme ). − DEB is by far the most genotoxic metabolite of BD, leading to chromosomal rearrangements such as sister chromatid exchanges (SCE) and exhibiting up to 200-fold higher mutagenic potency as compared to BD-derived monoepoxides. , DEB is also the active form of the antitumor drug treosulfan used for the treatment of ovarian cancer in Europe. − …”