Protein adducts: quantitative and qualitative aspects of their formation, analysis and applications.
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Proteins↗
Biomedical subjects
P Rydberg
Publications and source records attributed to P Rydberg.
A liquid chromatography tandem mass spectrometric method for in vivo dose monitoring of diepoxybutane, a metabolite of butadiene.
1,3-Butadiene, a common air pollutant formed in the combustion of organic matter, has been assessed by the U.S. EPA to be a strongly carcinogenic compound. This risk assessment is very uncertain because of the lack of information on the dose of the powerful carcinogenic metabolite diepoxybutane (DEB). This report presents an analytical method for in vivo dose monitoring of a unique marker for DEB. For a large number of alkylating agents in vivo doses are monitored by measurement by gas chromatography/mass spectrometry (GC/MS) of adducts to N-terminal valine in hemoglobin (Hb), using a modified Edman degradation method. This method is applicable to monofunctional epoxides from butadiene. However, in reaction with N-terminal valine, DEB forms an adduct which is ring-closed to a pyrrolidine, N,N-(2,3-dihydroxy-1,4-butadiyl)valine, with a tertiary amino group that prevents detachment of the alkylated valine by the Edman reagent. Therefore a method has been developed based on the analysis by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) of the N-modified N-terminal peptides enriched after trypsin digestion of globin. In this study Hb samples from mice injected intraperitoneally with (+/-)-DEB were examined qualitatively and quantitatively with regard to the ring-closed adduct. The N-terminal pyrrolidine-heptapeptide was identified in treated mice. The highest adduct levels were obtained in samples from animals given the highest dose of DEB and the adduct levels were below the detection level in control mice.
Alkylation↗
Heating of food and haemoglobin adducts from carcinogens: possible precursor role of glycidol.
Studies of adducts from reactive compounds to haemoglobin (Hb) by gas chromatography-tandem mass spectrometry according to the N-alkyl Edman method reveals the occurrence of N-(2,3-dihydroxypropyl)valine (diHOPrVal) at levels of 1-2 pmol/g Hb, in persons without known exposure. The hypothesis that this background originates from glycidol or related compounds during heating of food was tested in experiments with rats. Animals fed fried animal feed for 30 or 72 days showed an increase of the diHOPrVal level by about 50% compared with controls. Several arguments, such as the formation of reactive oxiranes by heat-induced dehydration of glycol configurations in glycerol and sugars, support the idea that glycidol (or e.g. glycidyl esters) are precursors of the adduct. In Hb samples, reduced for stabilisation of aldehyde adducts, relatively high levels of adducts determined as diHOPrVal were found, although without significant relation to frying of the feed. There is thus no indication that reduction in vivo of, for example, the Schiff base from glyceraldehyde, is a pathway for formation of the diHOPrVal. The background level of diHOPrVal in humans Hb is low, and the cancer risk associated with exposure to the specific alkylator-probably glycidol-formed in cooking, is therefore presumably low. The result implies, however, that low-molecular mass mutagenic oxiranes formed during the heating of food should be studied further.
Animals↗
Acrylamide: a cooking carcinogen?
Exposure to acrylamide (AA) has been monitored by mass spectrometric detection of the adduct, N-(2-carbamoylethyl)valine (CEV), to the N-termini of hemoglobin (Hb), according to the N-alkyl Edman method. In these studies, a conspicuous background level, about 40 pmol/g of globin, of apparently the same adduct was regularly observed in Hb from persons without known exposure to AA. For testing of the hypothesis that this adduct originates from AA formed in cooking, rats were fed fried animal standard diet for 1 or 2 months. These animals exhibited a strong increase of the level of the studied Hb adduct, compared to control rats fed unfried diet. By gas chromatography/tandem mass spectrometry, the identity with CEV was confirmed by the concordance of the product ion spectrum of the studied adduct with that of a verified standard and by interpretation of the fragment ions. Further support of the chemical structure, at the same time pinpointing AA as the causative reactive factor, was obtained through the demonstration that AA is formed in the heating of the feed and that the level of AA in the fried feed is compatible with the measured levels of the CEV adduct. The raised CEV adduct levels observed in experimental animals are of a magnitude that is similar to the background level in nonsmoking humans. These data render it likely that cooking of food is a major source of the background dose of AA also in humans. An evaluation of cancer tests of AA and available data for its metabolism leads to the estimation that the background dose of AA is associated with a considerable cancer risk.
Acrylamide↗
Adducts to N-terminal valines in hemoglobin from butadiene metabolites.
In order to identify a hemoglobin adduct useful for monitoring of doses of butadiene metabolites, particularly the strongly genotoxic, bifunctional diepoxybutane (DEB), the reaction of DEB with valinamide, a relevant model of globin N-termini, was studied. A preliminary kinetic analysis showed that the primary reaction product of DEB with valine-N gives, as was expected, rise to a ring-closed pyrrolidine-structured compound, N,N-(2,3-dihydroxybuta-1,4-diyl)valine (PYRV), in a reaction which is fast when compared to hydrolysis of the second oxirane ring with formation of N-(2,3,4-trihydroxybutyl)valine (THBV). The ring closure is also fast when compared to the rate of formation of a cross-linked divaline product. PYRV can therefore be used as a specific marker of in vivo doses of DEB whereas THBV may be applied for the dosimetry of the metabolite (1,2-dihydroxyethyl)oxirane. The latter is formed by half-hydrolysis of DEB or oxygenation of 1,2-dihydroxy-3-butene. The N-alkyl Edman method, used for specific cleavage and gas chromatographic/mass spectrometric (GC/MS) determination of adducts to N-terminal valine in hemoglobin, could be used for measurement of THBV, as shown in alkylation experiments with blood. However, the adduct specific for DEB, PYRV, requires-due to its tertiary amine structure-other techniques. The reaction products were identified by GC/MS, PYRV by 13C and 1H NMR, and THBV because of its formation by reduction of the Schiff bases of threose and erythrose with hemoglobin.
Alkylation↗