Discovery of Novel N-(4-Hydroxybenzyl)valine Hemoglobin Adducts in Human Blood

Abstract

Humans are exposed to a wide range of electrophilic compounds present in our diet and environment or formed endogenously as part of normal physiological processes. These electrophiles can modify nucleophilic sites of proteins and DNA to form covalent adducts. Recently, powerful untargeted adductomic approaches have been developed for systematic screening of these adducts in human blood. Our earlier untargeted adductomics study detected 19 unknown adducts to N-terminal valine in hemoglobin (Hb) in human blood. We now describe a full characterization of one of these adducts, which corresponds to the addition of a 4-hydroxybenzyl (4-OHBn) group to N-terminal valine in Hb to form N-(4-hydroxybenzyl)valine (4-OHBn-Val). The adduct structure was determined by comparison of its accurate mass, HPLC retention time, and MS/MS fragmentation to that of authentic standards prepared by chemical synthesis. Average 4-OHBn-Val adduct concentrations in 12 human blood samples were estimated to 380 ± 160 pmol/g Hb. Two possible routes of 4-OHBnVal adduct formation are proposed using two different precursor electrophiles: 4-quinone methide (4-QM) and 4-hydroxybenzaldehyde (4-OHBA). We found that 4-QM reacts rapidly with valine to form the 4-OHBn-Val adduct; however, the quinone methide is unstable under physiological conditions due to hydrolysis. It was shown that 4-OHBA forms reversible Schiff base adducts with valine, which can be stabilized via reduction in blood generating the 4-OHBn-Val adduct. In addition, trace amounts of isomeric 2-hydroxybenzyl-valine (2-OHBn-Val) adducts were detected in 12 human blood samples (estimated mean adduct level, 5.0 ± 1.4 pmol/g Hb). Further studies are needed to quantify the contributions from identified possible precursor electrophiles to the observed hydroxybenzyl adducts in humans.

Protein Adductomics: Methodologies for Untargeted Screening of Adducts to Serum Albumin and Hemoglobin in Human Blood Samples

“…The assumption that all FTH derivatives of Val adducts have similar responses in the MS analysis is a simplification but a reasonable approximation for the low molecular weight adducts observed. The supposed similar responses are supported by the comparable responses for the different known adducts that have been studied as FTH derivatives in more detail [48,49,50,51,52].…”

“…Formation of the Cys34 sulfenic acid, also leads to disulfide formation with small circulating thiols, which represent the largest class of adducts detected with HSA adductomics thus far. With Hb adductomics 25 different N-terminal Val adducts have been detected, with added masses ranging from 15 to 198 Da, with 13 of them identified thus far [19,50,52,57]. These Hb adducts are presented in Table 4.…”

“…Adducts from isocyanates, carbamoylated N-terminal valines, could though be detached as hydantoins from Hb after acidification in a procedure similar to the normal Edman procedure, as has been shown in several studies (e.g., for methylisocyanate [60] and dimethylformamide [61]). Of the identified or annotated adducts observed to Hb and HSA there has been very little overlap thus far (when analyzing blood samples from smokers, acrylonitrile, and ethylene oxide adducts were observed both to Hb and HSA [19,36], and the recently identified 4-hydroxybenzyl Hb adduct may correspond to the unknown HSA adduct with the same annotated composition C 7 H 7 O, added mass 107.0481 Da [52]). This emphasizes the need of several nucleophilic targets when exploring the adductome.…”

Internal Doses of Glycidol in Children and Estimation of Associated Cancer Risk

“…Both compounds, though, require reduction after formation of a Schiff base to the N-terminal valine in Hb to form the stable diHOPrVal adduct [30]. It is not known whether the reduction of protein adducts from Schiff bases occurs in vivo, but it was observed to occur in blood in vitro [31]. Epichlorohydrin, from occupational exposure, also could form diHOPrVal but it is not a probable exposure source in the presently studied group of humans [27].…”

Enhanced Tumor Selectivity of 5-Fluorouracil Using a Reactive Oxygen Species-Activated Prodrug Approach

“…Spontaneous breakdown of this intermediate will release 5FU, along with a highly reactive byproduct 4-methylenecyclohexa-2,5-dien-1-one, which is rapidly hydrolyzed in aqueous environment to give the nontoxic 4-(hydroxymethyl)phenol. 36 The synthesis of prodrugs 1a,1b and a control compound 1c is detailed in Scheme 1. The N1-selective benzylation of 5FU using either compound 2 or compound 3 in the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) as the base generated phenylboronate ester 1a and control 1c in modest yields.…”