Background: Acrylamide (AA) from diet in pregnancy has been associated with reduced birth weight (BW), but the impact on fetal size is unknown.

Objectives: To assess prenatal exposure to AA and associations with fetal size and size at birth.

Methods: Prenatal exposure to AA was measured using a high-throughput method evaluating hemoglobin adducts from AA (HbAA) and glycidamide (HbGA) in blood from pregnant women participating in the Danish Fetal Origins 1988-89 cohort (N = 991). Associations between HbAA and clinical records of fetal growth restriction (clinician-based FGR), biparietal diameter (BPD) at 16 gestational weeks, and small-for-gestational-age (SGA), BW, birth head circumference (BHC) and birth length (BL) were evaluated in the full population and after stratification by maternal smoking during pregnancy in multivariable models.

Results: Maternal HbAA median (5th-95th percentile) was 92 (45–264) pmol/g Hb and active smoking was common (42 %). Higher levels of HbAA were associated with non-significant smaller BPD. Compared to the lowest quartile, the highest quartile of HbAA was associated with a higher odds ratio of 4.00; 95 % confidence interval (CI; 1.23, 16.00) for clinician-based FGR and of 4.03 (95 %CI 1.46, 13.16) for SGA and a mean reduction in BW of 292 g (95 %CI -423, −161). Each 10-pmol/g Hb increase in HbAA was associated with a smaller offspring size at birth: 11 g (95 %CI -18, −5) for BW, −0.05 cm (−0.09, −0.01) for BHC, and −0.03 cm (−0.07, −0.01) for BL. These associations were evident after adjustment for smoking, but not in the smaller subsets of nonsmokers (n = 366) or subjects with HbGA measurement (n = 280).

Conclusion: This biomarker-based cohort study provides new evidence of an increase in the risk of clinician-based FGR, a critical indicator of long-term health, following prenatal exposure to AA. Furthermore, our findings add to the existing evidence that prenatal exposure to AA are associated with reduced newborn size at birth and call for more research on the effects of exposure to AA early in fetal growth and development.

Blood samples (n = 600) from participants in the Swedish dietary survey Riksmaten Adolescents 2016–17 were analyzed with respect to hemoglobin (Hb) adducts from acrylamide (AA) and its metabolite glycidamide (GA) as biomarkers of internal dose/exposure. The results are presented from statistical analyses of food consumption data (2-day dietary recall and questionnaires) and measured Hb adduct levels. The estimated exposure as well as consumption data were examined in relation to non-dietary factors such as sex, age (group medians of 12, 15, and 18 years), place of residence (urban/rural), smoking status, and parental education level. The median AA adduct level was estimated to be 34 pmol/g Hb (range 14–225). No significant difference was found for place of residence, parental education, sex, or age. A significant difference was found between the median adduct levels of daily smokers (n = 8) and never smokers (n = 323) in the older age groups, but not between occasional smokers (n = 47) and never smokers. The median differences between daily smokers and never smokers were 76, 40, and 128 pmol/g Hb for AA, GA, and AA + GA, respectively. The median AA intake for the whole group of adolescents, as estimated from dietary recall data combined with reported concentrations in food, was 0.40 μg/kg bw/day. The corresponding median intake estimated from measured Hb adduct levels of AA was 0.20 μg/kg bw/day. A significant, although low, positive Spearman correlation was found between the two intake estimates (p-value = 8 × 10−3; ρ = 0.11). From the estimated intake of AA from food frequency questionnaires, significance was found for the 15-year-old children with higher AA adduct levels observed at higher consumption frequencies of fried potatoes/French fries. AA is considered a genotoxic carcinogen. For the estimated intake of AA for any age group and method (dietary recall or AA adduct), both a calculated margin of exposure as well as lifetime quantitative cancer risk estimates indicate health concern. A future study on food consumption designed with respect to AA exposure would provide a better understanding of the correlation between consumption and exposure and should give a more reliable estimate of the contribution of dietary AA to the overall cancer risk.

Thiamine deficiency is a large contributor to reduced reproduction success among salmonids throughout the northern hemisphere. In Scandinavia, this reproduction disorder is known as M74; while in North America, it is known as early mortality syndrome (EMS). The disorder fluctuates in magnitude from year to year. During years with high prevalence of the disorder, salmonid hatcheries that stock various aquatic systems to maintain the population size experience difficulties filling their quotas without thiamine treatment of alevins. The disorder is monitored both by observing the survival rate and by measuring the thiamine content of prefertilized eggs in the hatcheries. Here, a simple extraction procedure is presented, which allows for quantitative determination of the various phosphorylated forms of thiamine using liquid chromatography mass spectrometry but also allows for extraction in 96 deep-well plates and measurement of the total thiamine content using fluorescence monitoring with a plate reader, following oxidation of thiamine to thiochrome. The latter procedure could also be integrated into a highly portal system where the thiochrome is determined using the DeNovix QFX analyzer. The newly developed extraction procedure and cleanup method for fluorescence measurement represent the most versatile and simple methods to date for monitoring of thiamine in salmonid eggs. The methods produced accurate and precise data with quantification limits below the limit where the deficiency causes 100% lethality.

Acrylamide is a probable human carcinogen with widespread exposure via food. The present study compared acrylamide intake measurements obtained from haemoglobin adduct levels and self-registered dietary consumption data in a group of 144 Norwegian healthy adults. Acrylamide adducts to N-terminal valine in haemoglobin were measured and used to estimate the intake via the internal dose approach which showed a median (interquartile range) of 0.24 (0.19–0.30) μg/kg bw/day. Data from weighed food records and food frequency questionnaires from the same individuals were used for probabilistic modelling of the intake of acrylamide. The median acrylamide intake was calculated to be 0.26 (0.16–0.39) and 0.30 (0.23–0.39) μg/kg bw/day, respectively from the two sources of self-registered dietary consumption data. Overall, a relatively good agreement was observed between the methods in pairwise comparison in Bland-Altman plots, with the methods disagreeing with 7% or less of the values. The intake estimates obtained with the two dietary consumption methods and one biomarker method are in line with earlier dietary estimates in the Norwegian population. The Margin of Exposure indicate a possible health risk concern from dietary acrylamide. This is the first study with a comparison in the same individuals of acrylamide intake estimates obtained with these methods.

Hemoglobin (Hb) adducts are widely used in human biomonitoring due to the high abundance of hemoglobin in human blood, its reactivity toward electrophiles, and adducted protein stability for up to 120 days. In the present paper, we compared three methods of analysis of hemoglobin adducts: mass spectrometry of derivatized N-terminal Val adducts, mass spectrometry of N-terminal adducted hemoglobin peptides, and limited proteolysis mass spectrometry . Blood from human donors was incubated with a selection of contact allergens and other electrophiles, after which hemoglobin was isolated and subjected to three analysis methods. We found that the FIRE method was able to detect and reliably quantify N-terminal adducts of acrylamide, acrylic acid, glycidic acid, and 2,3-epoxypropyl phenyl ether (PGE), but it was less efficient for 2-methyleneglutaronitrile (2-MGN) and failed to detect 1-chloro-2,4-dinitrobenzene (DNCB). By contrast, bottom-up proteomics was able to determine the presence of adducts from all six electrophiles at both the N-terminus and reactive hemoglobin side chains. Limited proteolysis mass spectrometry, studied for four contact allergens (three electrophiles and a metal salt), was able to determine the presence of covalent hemoglobin adducts with one of the three electrophiles (DNCB) and coordination complexation with the nickel salt. Together, these approaches represent complementary tools in the study of the hemoglobin adductome. 

We are exposed to many chemicals in our everyday life. Some of these chemicals could pose risks for our health. To reduce such risks, it is important to know what we are exposed to, how much, and how toxic the chemicals are.

This thesis focuses on development and novel applications of a method for identifying and quantifying adducts as biomarkers of exposure to electrophiles. Electrophiles are reactive and can be measured as their stable reaction products (adducts) with haemoglobin (Hb) using the FIRE procedure™. This method utilizes Fluorescein Isothiocyanate to selectively cleave the adducted (R) N-terminal valine in human Hb, with a modified Edman procedure.

The primary aim was to further develop the FIRE procedure for application to a larger number of human blood samples, and to decrease the amount of sample needed. To achieve this, the method was adapted to 96-well plates. Further improvements resulted in a method that uses ca. half of the material used in the original method and can be used to analyse 1000 samples in one to two months.

The newly developed version of the FIRE procedure was applied to analyse blood from 144 Norwegian adults (Paper I) and 600 Swedish adolescents (Paper II), to estimate exposure to the electrophile acrylamide. IARC has classified acrylamide as a probable human carcinogen. Diet is a major source of exposure to acrylamide in the general population, as it is formed in carbohydrate-rich food during high temperature processing. Acrylamide intake was calculated from Hb adduct levels and compared with intake estimates calculated from self-reported food consumption data obtained from dietary records and food frequency questionnaires, combined with data for acrylamide content in food. In the Norwegian study, acrylamide intake was estimated by probabilistic calculations of the two types of food consumption data, which resulted in that no large difference in the median estimate obtained by these methods was observed in comparison of estimated daily intake from Hb adduct data (0.24-0.30 µg/kg body weight). In both studies (Papers I, II), the calculated margin of exposure with regard to risk for cancer indicates that acrylamide intake from food is of concern in the studied populations.

In Paper III, an unknown adduct, observed in earlier FIRE adductomics work, was identified with the help of high resolution accurate mass spectrometry, a synthesized standard, and nuclear magnetic resonance spectroscopy. A strategy to trace the source of the adduct was evaluated, and the epoxide glycidic acid was confirmed as a possible precursor, by measurement of adduct formation rate in vitro in human blood.

Finally, in Paper IV the FIRE procedure was compared to bottom-up proteomics to study Hb adducts from acrylamide, acrylic acid, glycidic acid, 2,3-epoxypropyl phenyl ether, 2-methyleneglutaronitrile, and 1-chloro-2,4-dinitrobenzene (DNCB). Adducts from all electrophiles were identified with bottom-up proteomics, with Cys93 in the beta chain of Hb as the most reactive side chain. The FIRE procedure was inefficient to detect bulkier/electron-withdrawing adducts, as from the contact allergen DNCB.

In conclusion, FIRE has high detectability for Hb adducts, although it is not suitable for adducts from all electrophiles. It is a valuable tool to identify and measure adducts, and to estimate exposure/intake of electrophiles. The new version of FIRE would facilitate the application for exposure measurement in epidemiological studies.

Acrylamide forms in many commonly consumed foods. In animals, acrylamide causes tumors, neurotoxicity, developmental and reproductive effects. Acrylamide crosses the placenta and has been associated with restriction of intrauterine growth and certain cancers. The impact on human health is poorly understood and it is impossible to say what level of dietary exposure to acrylamide can be deemed safe as the assessment of exposure is un-certain. The determination of hemoglobin (Hb) adducts from acrylamide is increasingly being used to improve the exposure assessment of acrylamide. We aim to outline the literature on Hb adduct levels from acrylamide in humans and discuss methodological issues and research gaps. A total of 86 studies of 27,966 individuals from 19 countries were reviewed. Adduct levels were highest in occupationally exposed individuals and smokers. Levels ranged widely from 3 to 210 pmol/g Hb in non-smokers from the general population and this wide range sug-gests that dietary exposure to acrylamide varies largely. Non-smokers from the US and Canada had slightly higher levels as compared with non-smokers from elsewhere, but differences within studies were larger than between studies. Large studies with exposure assessment of acrylamide and related adduct forming compounds from diet during early-life are encouraged for the evaluation of health effects.

Analytical methods and tools for the characterization of the human exposome by untargeted mass spectrometry approaches are advancing rapidly. Adductomics methods have been developed for untargeted screening of short-lived electrophiles, in the form of adducts to proteins or DNA, in vivo. The identification of an adduct and its precursor electrophile in the blood is more complex than that of stable chemicals. The present work aims to illustrate procedures for the identification of an adduct to N-terminal valine in hemoglobin detected with adductomics, and pathways for the tracing of its precursor and possible exposure sources. Identification of the adduct proceeded via preparation and characterization of standards of adduct analytes. Possible precursor(s) and exposure sources were investigated by measurements in blood of adduct formation by precursors in vitro and adduct levels in vivo. The adduct was identified as hydroxypropanoic acid valine (HPA-Val) by verification with a synthesized reference. The HPA-Val was measured together with other adducts (from acrylamide, glycidamide, glycidol, and acrylic acid) in human blood (n = 51, schoolchildren). The HPA-Val levels ranged between 6 and 76 pmol/g hemoglobin. The analysis of reference samples from humans and rodents showed that the HPA-Val adduct was observed in all studied samples. No correlation of the HPA-Val level with the other studied adducts was observed in humans, nor was an increase in tobacco smokers observed. A small increase was observed in rodents exposed to glycidol. The formation of the HPA-Val adduct upon incubation of blood with glycidic acid (an epoxide) was shown. The relatively high adduct levels observed in vivo in relation to the measured reactivity of the epoxide, and the fact that the epoxide is not described as naturally occurring, suggest that glycidic acid is not the only precursor of the HPA-Val adduct identified in vivo. Another endogenous electrophile is suspected to contribute to the in vivo HPA-Val adduct level. 

The general population is exposed to the genotoxic carcinogen glycidol via food containing refined edible oils where glycidol is present in the form of fatty acid esters. In this study, internal (in vivo) doses of glycidol were determined in a cohort of 50 children and in a reference group of 12 adults (non-smokers and smokers). The lifetime in vivo doses and intakes of glycidol were calculated from the levels of the hemoglobin (Hb) adduct N-(2,3-dihydroxypropyl)valine in blood samples from the subjects, demonstrating a fivefold variation between the children. The estimated mean intake (1.4 mu g/kg/day) was about two times higher, compared to the estimated intake for children by the European Food Safety Authority. The data from adults indicate that the non-smoking and smoking subjects are exposed to about the same or higher levels compared to the children, respectively. The estimated lifetime cancer risk (200/10(5)) was calculated by a multiplicative risk model from the lifetime in vivo doses of glycidol in the children, and exceeds what is considered to be an acceptable cancer risk. The results emphasize the importance to further clarify exposure to glycidol and other possible precursors that could give a contribution to the observed adduct levels.