Estimation of exposure to dietary acrylamide based

Journal of Exposure Science and Environmental Epidemiology (2015), 1–8 © 2015 Nature America, Inc. All rights reserved 1559-0631/15

www.nature.com/jes

ORIGINAL ARTICLE

Estimation of exposure to dietary acrylamide based on mercapturic acids level in urine of Polish women post partum and an assessment of health risk Hanna Mojska1, Iwona Gieleciń ska1, Aleksandra Zieliń ska2, Joanna Winiarek2 and Włodzimierz Sawicki2 We determined metabolites of acrylamide and glycidamide concentrations (AAMA and GAMA, respectively) in urine of 93 women within the first days after delivery, using LC-MS/MS. The median AAMA and GAMA levels in urine were 20.9 μg/l (2.3÷399.0 μg/l) and 8.6 μg/l (1.3÷85.0 μg/l), respectively. In smokers we found significantly (Po 0.01) higher levels of metabolites in comparison with the non-smoking women. As demonstrated by the 24-h dietary recall, acrylamide intake was low (median: 7.04 μg/day). Estimated exposure to acrylamide based on AAMA and GAMA levels in the whole group of women was 0.16 μg/kg b.w./day (1.15 μg/kg b.w./day, P95). We found significantly (Po 0.05) higher exposure in women who consumed higher amount of acrylamide in the diet (≥10 μg/day vs o10 μg/day). A weak but significant positive correlation between acrylamide intake calculated on the basis of urinary levels of AAMA and GAMA and estimated on the basis of 24-h dietary recall (r = 0.26, P o 0.05) was found. The estimated margin of exposure values were below 10 000 and ranged from 156 for 95th percentile to 1938 for median acrylamide intake. Our results have shown that even a low dietary acrylamide intake may be associated with health risk. Journal of Exposure Science and Environmental Epidemiology advance online publication, 1 April 2015; doi:10.1038/jes.2015.12 Keywords: acrylamide; urinary metabolites; dietary exposure; Polish women post partum; risk assessment; LC-MS/MS

INTRODUCTION Acrylamide (AA) is a chemical compound extensively used in the industry for the production of polyacrylamide polymers. They are used as flocculants to clarify drinking and industrial water, as gels for electrophoretic separation of proteins, in paper, cosmetic and textile industries. Increased interest in acrylamide has been noted since 2002, when the Swedish National Food Agency in collaboration with scientists from the Stockholm University1 published for the first time data on the high content of acrylamide in highcarbohydrate products undergoing thermal processing. Acrylamide forms in food mainly as a result of the Maillard reaction between free asparagine and reducing sugars. The factors that have an important role in acrylamide formation in food is the thermal processing temperature (4120 °C), a low moisture content of the product and an inactive matrix.2,3 The main source of acrylamide in the human diet are potato products, such as French fries and potato crisps, cereal products, such as bread, breakfast cereals, cookies, and also coffee and coffee substitutes. Acrylamide is also a component of tobacco smoke,4 and cigarette smoking is an important source of exposure to this compound.5,6 Acrylamide is a neurotoxic compound and may contribute to central and peripheral nervous system damage, both in laboratory animals and in humans exposed to this compound at the workplace.7,8 In animal studies, acrylamide administration in drinking water was associated with an increased incidence of cancer of

many organs, including testes, heart, uterus, adrenals, thyroid, mammary gland, oral cavity and skin.9,10 Although carcinogenic action of acrylamide was demonstrated in animal studies, the results obtained in epidemiological studies in humans are inconclusive. In numerous studies,11–13 no association was found between acrylamide intake with the diet and the development and occurrence of neoplastic tumours of various organs in humans. In other studies,14–17 a correlation was shown between an increase in the level of acrylamide adducts with haemoglobin in humans, resulting from dietary intake of acrylamide, and an increase in the risk of development of cancer of various organs. Already in 1994, the International Agency for Research on Cancer classified acrylamide in the group of compounds “probably carcinogenic to humans” (Group 2A),18 considering that although the evidence for the carcinogenic action of acrylamide in humans is limited, that action was well documented in laboratory animal studies. Considering that acrylamide is a genotoxic and a carcinogenic compound, the margin of exposure (MOE) criterium is recommended for its risk assessment.19 The MOE is the ratio of Benchmark Dose Lower Limit (BMDL10) to the estimated human intake of the compound. The BMDL10 is the lower bound of a 95% confidence interval on the benchmark dose (BMD) corresponding to a 10% tumour incidence. In 2011, the Joint FAO/WHO Expert Committee on Food Additives (JECFA) calculated MOE values for

1 Department of Food and Food Supplements, National Food and Nutrition Institute, Warsaw, Poland and 2Clinic of Obstetrics, Gynaecology and Oncology, 2nd Faculty of Medicine, Medical University of Warsaw, Warsaw, Poland. Correspondence: Dr. Hanna Mojska, Department of Food and Food Supplements, National Food and Nutrition Institute, 61/63 Powsińska Street, 02-903 Warsaw, Poland. Tel.: +48 22 55 09 656. Fax: +48 22 55 09 887. E-mail: [email protected] Received 4 November 2014; revised 12 January 2015; accepted 14 January 2015

Estimation of exposure to AA of women post partum Mojska et al

2 acrylamide using two different values of BMDL10: 0.31 mg/kg b.w./day and 0.18 mg/kg b.w./day based on the induction of mammary tumours in rats and Hardenrian gland tumours in mice, respectively. JECFA concluded that the margins of exposure for average and deep consumers of acrylamide were low for the compound that is genotoxic and carcinogenic and this might indicate to a human health concern.20 Acrylamide is rapidly absorbed and, owing to its very good solubility in water, rapidly distributed to various tissues. It is metabolised through two main metabolic pathways: epoxidation to glycidamide and glutathione conjugation to mercapturic acids.21 The conversion of acrylamide to glycidamide, its main metabolite, is catalysed by an enzyme of cytochrome P450 (isoenzyme CYP2E1).22 Both acrylamide and glycidamide form adducts with haemoglobin. Acrylamide adducts with N-terminal valine in the haemoglobin molecule (AA-Hg) are considered as biomarkers for the long-term exposure to acrylamide. Higher levels of AA-Hg were found in smokers and persons exposed to acrylamide at work.6,23–25 In addition, glycidamide forms a number of DNA adducts, this is why glycidamide is considered to have a critical role in the carcinogenic action of acrylamide.26 Both the compounds are conjugated with glutathione and further metabolised to form mercapturic acids: acrylamide to N-acetyl-S-(2-carbamoylethyl)-L-cysteine (AAMA) and glycidamide to N-acetyl-S-(2-carbamoyl-2-hydroxyethyl)-L-cysteine (GAMA) and N-acetyl-S-(3-amino-2-hydroxy-3-oxopropyl)-cysteine. Acrylamide and glycidamide metabolites in the form of mercapturic acids are excreted in the urine.27 Several papers published so far24,25,28–31 presented the results of studies on urinary levels of mercapturic derivatives of acrylamide and glycidamide in humans. The possibility of the use of mercapturic derivatives levels for the assessment of exposure to acrylamide from food and tobacco smoke was also presented. However, none of the studies so far presented the impact of a similar type of a diet low in acrylamide on AAMA and GAMA concentration in urine. In our previous study,32 the exposure to dietary acrylamide for the Polish population was estimated in a probabilistic approach on the basis of the 24-h recall. Here we present the results of estimation of exposure to acrylamide for the women, within the first days after delivery in the course of hospital stay, based on the urinary levels of AAMA and GAMA and health risk evaluation connected with dietary exposure to acrylamide using the MOE approach. Correlation between exposure to acrylamide calculated on the basis of AAMA and GAMA levels in urine and on the basis of the 24-h recall was also tested.

after the night) into a polypropylene container. The urine samples were temporarily stored in the hospital refrigerator and as soon as possible transported in ice to the laboratory in the National Food and Nutrition Institute where they were frozen and stored at − 70 °C until the analysis. On the same day on which morning the urine sample was collected, a face-to-face interview was held about food consumption within the past 24 h. All 24-h dietary recall were conducted by two trained persons (dieticians). The portion size was verified with the use of an ‘Album of Photographs of Food Products and Dishes’.33

Determination of Urinary Levels of AAMA and GAMA Chemicals. AAMA (N-acetyl-S-(2-carbamoylethyl)-L-cysteine) and d4-AAMA (N-acetyl-S-(2-carbamoylethyl-d4)-L-cysteine) were supplied by C/D/N Isotopes, Quebec, Canada, GAMA (N-acetyl-S-(2-carbamoyl-2-hydroxyethyl)L-cysteine dicyclohexyl-ammonium salt) and d3-GAMA (N-acetyl-S-(2-carbamoyl-2-hydroxyethyl)-L-cysteine-d3 dicyclohexyl-ammonium salt) were supplied by Toronto Research Chemicals, North York, Canada. Methanol HPLC (99.9%) was supplied by Rathburn Chemicals (Walkerburn, Scotland), acetonitrile LC-MS (99.9+) and methanol LC-MS (99.8+) were supplied by Avantor Performance Materials B.V. (Deventer, Netherlands), formic acid LC-MS (98%) was supplied by Fluka (Germany), ammonium formate (HPLC, 99.0+%) was supplied by Fluka (Switzerland) and formic acid 98–100% GR (ACS, Reag.) was supplied by Merck KGaA (Darmstadt, Germany). ISOLUTE ENV+ (100 mg; 10 ml) columns supplied by Biotage AB (Uppsala, Sweden) were used for solid-phase extraction (SPE). Stock reference solutions of AAMA and d4-AAMA were prepared by dissolving 10 mg of each substance in 10 ml of methanol (LC-MS), whereas the stock reference solutions of GAMA and d3-GAMA were prepared by dissolving 5 mg of each substance in 10 ml of methanol; the solutions obtained were diluted with the same solvent to 100 mg/l and 10 mg/l, respectively. A mixture of reference solutions (of d4-AAMA and d3-GAMA) at a concentration of 10 mg/l was added to all samples. All reference solutions were stored at ~ − 20 °C in amber glass vials.

Sample Preparation of Urine The samples were prepared according to Boettcher and Angerer.5 In brief, after thawing to room temperature and mixing, 4 ml of urine were withdrawn from each sample and 30 μl of the mixture of d3- and d4internal standard solutions were added. The samples were vortex mixed, centrifuged and then purified on SPE columns. The supernatant was applied to the columns that were previously conditioned with methanol, water and diluted formic acid. The tested compounds were eluted with 1% formic acid solution in methanol for HPLC. After evaporation to dryness, the collected eluate was dissolved in 0.5 ml of 0.1% formic acid (LC-MS) solution (v/v) and analysed by LC-MS/MS.

LC-MS/MS Analysis

The study group was recruited among healthy women who gave birth at term to healthy babies in the obstetric ward of the Clinic of Obstetric, Gynaecology and Oncology in Medical University of Warsaw between January and March 2012. After explaining the purpose of the research 110 women granted a written consent to participate in the study. They were asked to complete a sociodemographic survey that include questions about age, education, place of residence and smoking habits. Nevertheless, 5 participants did not provide the urine sample, and the 24-h dietary recall was not conducted in 12 other women. Therefore, the final study group consisted of 93 subjects. Women who participating in our study stayed in the hospital for 2–5 days depending on their health status and/or health status of their child. In all women, measurements of actual weight were performed. The Ethics Committee at the National Food and Nutrition Institute in Warsaw approved the study.

Chromatographic separation was conducted on the Kinetex 2.6 u XB-C18 column supplied by Phenomenex, with the use of UltiMate 3000 liquid chromatograph (LC) supplied by Dionex. Analysis conditions: flow rate – 1 ml/min, column temperature − 40 °C, injection volume − 10 μl, mobile phase–water and acetonitrile LC-MS (96: 4) with an addition of 0.2% formic acid and 2 mM ammonium formate; runtime – 1.5 min. Samples were eluted isocratically. AAMA/ d4-AAMA and GAMA/ d3-GAMA were eluted at 0.42 and 0.34 min, respectively. A mass spectrometer (MS-MS) 3200 QTrap supplied by ABSciex was used for the assays of acrylamide and glycidamide metabolites. The analysis was performed by MRM (multiple reaction monitoring) in negative polarity. Conditions of spectrometric analysis: curtain gas – nitrogen (CUR = 30), ion source temperature – 600 °C, electrospray capillary voltage (IS) – of − 4.500 V, dwell-time – 50 ms. The collision energy (CE) is stated between brackets at the individual ions. Ions of the tested compounds: m/z 233 → 104 (AAMA, CE: − 22), m/z 236.9 → 108 (d4-AAMA, CE: − 22), m/z 248.9 → 120.1 (GAMA, CE: − 24) and m/z 252 → 119.9 (d3-GAMA, CE: − 24) were monitored for the purposes of quantitative determination of AAMA and GAMA levels in the urine, whereas m/z 233 → 58 (AAMA, CE: − 54) and m/z 248.9 → 127.9 (GAMA, CE: − 18) were monitored for the purposes of verification of the results obtained.

Urine Sampling and Conducting the 24-h Dietary Recall

Calibration Procedure and Quality Controls

On day 2 to 5 after birth, depending on the length of hospital stay, each of the participants collected the morning urine sample (the first passed urine

The limit of quantification (LOQ) was determined by using a signal-to-noise (S/N) ratio of 10. The S/N ratio calculation was performed using the scripts:

MATERIALS AND METHODS Study Group

Journal of Exposure Science and Environmental Epidemiology (2015), 1 – 8

© 2015 Nature America, Inc.


3 Savitzky-Goolay Smooth and Signal-to-Noise (Applied Biosystems). The eight-point calibration curve was plotted for the individual compounds within a range of 1–500 μg/l at a constant level of d4-AAMA and d3-GAMA (c = 75 μg/l), taking into account the anticipated levels of the studied compounds in the urine. The calibration curve was constructed by plotting the amount, in micrograms, of AAMA and GAMA an the corresponding d3- and d4-labelled standards against the response (area) of the target ion. To verify the stability of the calibration curves one low- and one highconcentration mix standard solution and one blanc was included in each analytical series. The LC-MS/MS method of measurement of urinary levels of AAMA and GAMA was validated. The following parameters were determined: selectivity, limit of quantification, method range, precision, repeatability and accuracy by recovery testing.

Determination of Urinary Creatinine Levels Urinary creatinine levels were measured by the VITROS CREA Slide method, which is performed using the VITROS CREA Slides and the VITROS Chemistry Products Calibrator Kit 1 on the VITROS-350 biochemistry analyser supplied by Ortho-Clinical Diagnostics (USA).34

Estimation of Daily Acrylamide Intake on the Basis of Urinary Levels of AAMA and GAMA Acrylamide intake (NAA; μg/kg b.w./day) based on the measured urinary levels of AAMA and GAMA was calculated by the following formula:29 NAA ¼ ½ðsum of AAMA þ GAMAÞ ´ KW ´ MAA =½0:5 ´ MAAMAþGAMA where: sum of AAMA+GAMA (μg/g creatinine); KW-creatinine excretion (mg/kg b.w./day). The actual creatinine contents in the urine of each study subject determined analytically, the study subject’s weight, and the average amount of urine excreted daily by an adult woman were taken into account in the calculation of urinary excretion of creatinine. In accordance with the Geigy table,35 it was assumed that an adult woman excreted 1.4 l of urine per day; MAA – molar mass of acrylamide (MAA = 71); 0.5 – conversion factor; adopted from Boettcher et al.36 MAAMA+GAMA – sum of molar masses of metabolites: AAMA and GAMA (MAAMA+GAMA = 476).

Assessment of Dietary Acrylamide Intake Based on Data from Dietary Recall The dietary intake of acrylamide was calculated by taking into account our earlier own data on acrylamide content in food products in Poland.32,37–39 Acrylamide content in each product consumed by the study subjects (in μg/kg) was multiplied by the size of portion of that product (in grams) estimated on the basis of the 24-h dietary recall, and the sum of the daily intake of acrylamide from all consumed products was then divided by the body weight individually for each woman participating in the study (in kilograms). Ten women received only a drip infusion after delivery. These were Ringeri solution, 0.9% NaCl, 5% glucose or multi-electrolytes solutions supplied by Fresenius Kabi, Poland. Measured acrylamide content in these solutions was below the limit of quantification (0.1 μg/l).

Statistical Analysis The results of urinary levels of AAMA and GAMA expressed as a median and range (min-max) are presented in μg/l urine and in μg/g creatinine. The dietary intake of acrylamide is presented in μg/person/day and μg/kg b.w./day. Non-parametric tests: Mann–Whitney U-test was used for the comparison between the study groups. Spearman's correlation analysis was performed to determine the bivariate correlation between the exposure calculated on the basis of the level of AAMA and GAMA in the urine and acrylamide intake from the diet estimated on the basis of the 24-h dietary recall. P-value o 0.05 was considered significant for the significance of differences or dependent correlation. Statistical analyses were carried out with the use of Statistica ver. 6.0. © 2015 Nature America, Inc.

RESULTS Characteristics of Women Table 1 presents the characteristic of women participating in our study. Most of them have university degrees and lived in Warsaw. Five of the study subjects declared having smoked cigarettes throughout their pregnancy and 10 more were exposed to tobacco smoke at home. Dietary Acrylamide Intake The hospital diet consumed by the women participating in the study consisted mainly of milk and milk products, cooked and stewed meat and processed meat, cooked potatoes and vegetables, rice and cereal products in the form of bread, bread rolls, cereal flakes. In addition, the study subjects consumed various cookies, rusks, chocolate and chocolate products. The beverages were mainly water, tea, fruit juices and chicory coffee. On the basis of the acrylamide contents in food and the declared consumption of food products and dishes we found that the main source of acrylamide in the diet of the participants was soft bread (70.4%), followed by various cookies and cakes (24.8%). The remaining 4.8% was delivered, in the decreasing order, by crispbread, chocolate and chocolate products, chicory coffee and cereal flakes. Table 2 shows the median of dietary acrylamide intake during 24 h period before urine sample collection. For the 10 women, who received only a drip infusion after delivery, the dietary intake of acrylamide was assumed as 0 μg/person/day. In the remaining group of 78 persons, acrylamide intake at a level of 10 μg/day was adopted as the limit value for the division. In a group of 88 women the median dietary intake of acrylamide estimated on the basis of 24-h dietary recall was 7.04 μg/day. Significant (P o 0.000005) higher level was found in the group of women consuming ≥ 10 μg acrylamide per day compared with the other groups. Validation of the LC-MS/MS Method It was found that the LC-MS/MS method is suitable for analysing urinary AAMA and GAMA concentration and enabled the correct separation of the tested compounds from other matrix ingredients and obtaining daughter ions characteristic for AAMA and GAMA as well as their deuterated forms, and thus was specific for the compounds tested. The limit of quantification was calculated as 1 μg/l for both the analytes. All calibration curves were linear within the given

Table 1.

Characteristics of participants.

Variable Age (years; n = 93) Weight (kg; n = 93) Creatinine concentration (μg/l; n = 93)

Mean (range) or number of persons (%) 30 (20–40) 73 (51–111) 788 (77–2893)

Education level: Elementary and secondary school High school University

5 (5.4) 26 (28.0) 62 (66.6)

Place of residence: Town Village

81 (87.1) 12 (12.9)

Smoking habits during pregnancy: Smokers Passive smokers Non-smokers

5 (5.4) 10 (10.8) 78 (83.8)



4 Table 2.

Dietary acrylamide intake based on the 24-h dietary recall.

Group

Acrylamide intake (μg/day) Median (min–max)

Total (n = 93) Smokers (n = 5) Passive smokers (n = 10) Non-smokers (n = 78) Total (n = 88)a Drip infusion (n = 10) Acrylamide intake o10 μg/day (n = 55) Acrylamide intake ≥ 10 μg/day (n = 23)

7.03 4.83 4.52 7.16

(0.0–51.3) (0.0–18.0) (0.0–51.3) (0.0–49.6)

7.04 (0.0–51.3) 0.00b 6.05b (1.55–9.95) 15.03b (10.7–51.3)

(μg/kg b.w./day) P95

Median (min–max)

P95

25.1 18.0 51.3 21.8

0.10 0.06 0.06 0.11

(0.00–0.66) (0.00–0.24) (0.00–0.59) (0.00–0.66)

0.38 0.24 0.59 0.32

25.1 — 9.7 49.6

0.10 (0.00–0.66) 0.00b 0.09b (0.03–0.15) 0.23b (0.11–0.66)

0.38 — 0.14 0.60

a

Smoker’s excluded, passive smokers included. bStatistically significant difference depending on the dietary intake of acrylamide between all compared group (Po0.000005).

concentration range (1–500 μg/l) and linear correlation coefficients were 40.999 (AAMA: r = 0.9993, n = 3; GAMA: r = 0.9994, n = 3). The following validation parameters were obtained within the entire range of both curves: AAMA – within-day precision: RSD (coefficient of variation) o 3.8%, bias ± 5.4%, n = 3; between-day precision: RSD o 6.6%; bias ± 3.1%, n = 6; GAMA – within-day precision: RSD o 5.3%, bias ± 5.9%, n = 3; between-day precision: RSD o8.2%; bias ± 3.7%, n = 6. The precision of the method was determined by analysing six parallel urine samples, at two different levels of content of the tested compounds. The coefficients of variation were 2.9% (11 μg/l) and 3.2% (145 μg/l) for AAMA and 5.4% (6 μg/l) and 4.9% (21 μg/l) for GAMA. The accuracy of the method was verified on the basis of an analysis of recovery of the tested compound. The mean recovery for AAMA was 101.8% and for GAMA was 103.9%. The obtained validation results indicate that the LC-MS/MS method of AAMA and GAMA assay in urine is specific for the tested compound and is characterised by both good precision and accuracy. Urinary Levels of AAMA and GAMA Table 3 presents AAMA and GAMA concentration in urine of participants. In all urine samples, the AAMA and GAMA levels were above the limit of quantification. The median levels of AAMA and GAMA in the whole group of 93 study subjects reached 20.9 μg/l urine (31.8 μg/g creatinine) and 8.6 μg/l urine (12.2 μg/g creatinine), respectively. Values ranged widely from 2.3 to 399 μg/l (7.8–382.2 μg/g creatinine) for AAMA and from 1.3 to 85.0 μg/l (4.9–68.5 μg/g creatinine) for GAMA. The average level of AAMA in the urine of smokers was over eight times higher than in the group of non-smokers and passive smokers (P o0.05). A similarly significantly (P o 0.01) higher level of GAMA was found in the urine of smokers in comparison with non-smokers and women passively exposed to tobacco smoke. We also found a strong correlation between AAMA and GAMA both for the whole group of 93 women (r = 0.91, P o 0.00001) as well as for the non-smoking group (r = 0.90, P o 0.00001) and the group of passive smokers (r = 0.94, Po 0.0001). To ensure uniformity of the group, smoking women were excluded from further investigations on the effect of diet on urinary levels of AAMA and GAMA. As we did not find any effect of passive smoking on urinary levels of mercapturic derivatives of acrylamide and glycidamide, we performed the further assessment in the combined group of 88 women who were nonsmokers and passive smokers. The results are presented in Table 4. In the group of women whose dietary intake of acrylamide was ≥ 10 μg/person, a significantly (P o 0.05) twice higher AAMA level Journal of Exposure Science and Environmental Epidemiology (2015), 1 – 8

was found in comparison with the two remaining groups. The urinary levels of GAMA did not differ significantly among the above three groups of women. Estimates of Daily Exposure to Acrylamide and Health Risk Assessment Table 5 presents the estimated acrylamide intake in the study subjects on the basis of urinary levels of AAMA and GAMA. For the whole group of 93 women, the median acrylamide intake was 0.16 μg/kg b.w./day, attaining a value of 1.15 μg/kg b. w./day at the 95th percentile. In the group of smoking women, exposure to acrylamide was nine times significantly (P o0.00001) higher than that in non-smoking and passive smoking. A significantly (P o 0.05) higher exposure to acrylamide was also found in the group of women consuming ≥ 10 μg of acrylamide per day in comparison with the remaining two groups. The estimated MOE values, in the whole group of women participating in our study, ranged from 1125 to 1938 (median) and from 156 to 269 (P95). About ten lower values of MOE we found in smokers. We found a weak but significant positive correlation between acrylamide intake estimated on the basis of urinary metabolites and on the basis of 24-h dietary recall for the whole group of 93 women (r = 0.26, P o 0.05; Figure 1), and also in the group of 88 non-smoking women (r = 0.31, P o0.005; Figure 2). DISCUSSION In present study, we analysed the content of metabolites of acrylamide and glycidamide in urine of women at the period after the physiological fasting (after delivery). Our results were used to assess acrylamide exposure in a group of women with a low dietary acrylamide intake. Also they were used to assess the effect of a diet type (hospital diet) on the level of AAMA and GAMA in the urine of women within the first days after delivery. Determination of AAMA and GAMA in urine was performed by the LC-MS/MS method. The results of the validation confirm that the developed method is characterised by good selectivity, precision, repeatability and accuracy. In all the tested urine samples, the AAMA and GAMA levels were above the limit of quantification (1 μg/l). The median urinary level of AAMA was 20.9 μg/l and GAMA was 8.8 μg/l with values ranging from 2.2 to 399 μg/l and 1.3 to 85 μg/l, respectively. The median urinary level of AAMA in all subjects in present study was several times lower compared with the results obtained in adults by other authors.6,25,28 This is self-evident in view of the fact that participating women in our study were in the period of the first © 2015 Nature America, Inc.


5 Table 3.

Levels of AAMA and GAMA in the urine of study subjects, taking into account exposure to tobacco smoke.

Parameter

Total (n = 93) Median

Active smokers (n = 5)

Passive smokers (n = 10)

Min—max

Median

Min—max

2.3–399 1.3–85 3.7–471 0.13–1.01

168a 26.7a 190a 0.18

58.4–399 22.1–85 85.1–471 0.13–0.46

21.6 9.4 31.0 0.41

On creatinine basis (μg/g creatinine) AAMA 31.8 7.8–382 GAMA 12.2 4.9–68.5 AAMA+GAMA 44.8 15.0–451

165a 47.4a 214a

54.9–382 15.1–68.5 80.1–451

25.2a 10.3a 35.4a

On volume basis (μg/l) AAMA GAMA AAMA+GAMA GAMA: AAMA

20.9 8.6 29.2 0.37

Median

Min—max

Non-smokers (n = 78)

P-value

Median

Min–max

9.0–73.9 3.5–30.9 14.0–105 0.26–0.74

18.9 6.8 26.8 0.37b

2.3–148 1.3–50.7 3.7–181 0.13–1.01

o0.005 o0.01 o0.005 o0.05

8.6–123 6.4–51.3 15.0–174

31.4a 12.2a 44.2a

7.8–138 4.9–51.3 15.0–174

o0.01 o0.05 o0.005

Abbreviations: AAMA, N-acetyl-S-(2-carbamoylethyl)-L-cysteine; GAMA, N-acetyl-S-(2-carbamoyl-2-hydroxyethyl)-L-cysteine. Statistical significances: acompared with other groups (Passive smokers; Non-smokers), bcompared with group of Active smokers.

Table 4.

Levels of AAMA and GAMA in the urine of study subjects, taking into account the dietary intake of acrylamide.a

Parameter Total (n = 88) On volume basis (μg/l) AAMA GAMA AAMA+GAMA GAMA: AAMA

P-value

Urinary levels of AAMA and GAMA

19.6 8.2 27.0 0.37

(2.3–148) (1.3–50.7) (3.7–181) (0.13–1.01)

On creatinine basis (μg/g creatinine) AAMA 30.7 (7.8–138) GAMA 11.8 (4.9–51.3) AAMA+GAMA 43.3 (15.0–174)

Drip infusion (n = 10)

12.8 8.1 21.8 0.54

(3.7–27.1) (2.6–11.8) (6.3–38) (0.40–1.01)

20.3 (7.8–36) 10.4 (4.9–17.1) 31.7 (15.0–50.9)

Acrylamide intake o10 μg/day (n = 55)

Acrylamide intake ≥ 10 μg/day (n = 23)

(2.3–114) (1.3–50.7) (3.7–165) (0.21–0.76)

29.20b (6.1–148) 9.7 (2.5–33) 36.0c (8.5–181) 0.25d (0.13–0.50)

o 0.05 NS o 0.05 o0.005

28.1 (14.2–111) 10.8 (4.9–33.4) 38.0 (20.8–144)

55.1d (23.8–138) 13.7 (6.8–51.3) 70.9d (30.9–174.)

o 0.05 NS o 0.05

18.3 7.2 23.3 0.38

Abbreviations: AAMA, N-acetyl-S-(2-carbamoylethyl)-L-cysteine; GAMA, N-acetyl-S-(2-carbamoyl-2 hydroxyethyl)-L-cysteine; NS, not significant. aEstimated on the basis of the 24-h dietary recall. The results are presented as a median. The values between brackets are the minimum and maximum. Statistical significances: b compared with other groups (Drip infusion; Acrylamide intake o10 μg/day), ccompared with group of Drip infusion, dbetween all compared groups.

days after giving birth and consumed small quantities of food and, as demonstrated by the 24-h dietary recall, dietary acrylamide intake was low (median: 7,04 μg/day). In addition, about 11% of 93 study subjects received only a drip infusion after delivery. The average level of GAMA in urine of all women participating in present study was ~ 2.5 times lower in comparison with AAMA. It should be noted that despite the different diet, urinary level of GAMA in our study and in the study of Boettcher et al.25 was very similar, 8 μg/l (total group) and 5 μg/l (non-smoking group), respectively. Also in a group of 60 adult non-smokers in Germany6 the median level of GAMA was similar (8.7 μg/l). A slightly lower average urinary level of GAMA (3 μg/l) was found by Bjellaas et al.28 in 47 non-smokers in Norway. The presented results seem to indicate the limited conversion of dietary acrylamide to glycidamide in humans and its limited further conversions to GAMA. This requires further studies. It turned out that in the study group of 93 subjects, 5 women smoked cigarettes throughout their pregnancy and another 10 were passively exposed to tobacco smoke. Despite the small size of these groups, we decided to assess the effect of tobacco smoking on the urinary levels of the tested compounds. In the group of actively smoking women, the median urinary levels of AAMA (168 μg/l) and GAMA (26.7 μg/l) were significantly (P o 0.005 and P o 0.01, respectively) higher compared with non-smoking and passive smoking subjects. Also the other authors found significantly higher urinary levels of mercapturic © 2015 Nature America, Inc.

derivatives of acrylamide and glycidamide in smokers.6,25,28 The results obtained by us seem to confirm that cigarette smoking is an important source of exposure to acrylamide. Similarly as found by Heudorf et al.29 in a group of 110 children in Germany and Brisson et al.31 in group of 195 Canadian teenagers, in our study we did not find any significant differences in the median of urinary levels of AAMA and GAMA in non-smoking women and women passively smoking in the course of pregnancy. Opposite results were obtained by Ji et al.30 in a group of 39 children in Korea. Nevertheless, the lack of differences between non-smokers and passive smokers found in our study may result from the fact that women participating in our study were not exposed to tobacco smoke during their stay in the hospital. We also found a strong correlation between AAMA and GAMA both for the whole group of 93 women (r = 0.91, P o 0.00001) as well as for the non-smoking group (r = 0.90, P o 0.00001) and the group of passive smokers (r = 0.94, P o 0.0001). Our results seem to confirmed that acrylamide is the parent compound for both the metabolites: AAMA and GAMA, in the urine. In our study, we intended to assess the effect of one diet type (hospital diet) delivering a relatively small amount of acrylamide on urinary levels of AAMA and GAMA and to assess the dietary exposure to acrylamide in a group of women at the period after giving birth (physiological fasting). To ensure uniformity of the group, smoking women were excluded from further investigations. As we did not find any effect of passive smoking on the Journal of Exposure Science and Environmental Epidemiology (2015), 1 – 8


6 Table 5.

Acrylamide exposure estimated on the basis of urinary levels of AAMA and GAMA and MOE values.

Group

Acrylamide exposure (μg/kg b.w./day)

MOE (BMDL10 = 0,18 mg/kg b.w./day)

Median (min–max)

P95

Total (n = 93) Smokers (n = 5) Non-smokers (n = 88)

0.16 (0.02–2.62) 1.35a (0.41–2.62) 0.15a (0.02–1.40)

1.15 2.62 0.59

1125 133 1200

156 69 305

1938 229 2067

269 118 525

Total (n = 88)b Drip infusion (n = 10) Acrylamide intake o10 μg/day (n = 55) Acrylamide intake ≥ 10 μg/day (n = 23)

0.15 0.12 0.15 0.22c

0.59 0.24 0.64 0.59

1200 1500 1200 818

305 750 281 305

2067 2583 2067 1409

525 1292 484 525

(0.02–1.40) (0.05–0.24) (0.02–0.98) (0.06–1.40)

Median

P95

MOE (BMDL10 = 0,31 mg/kg b.w./day) Median

P95

2.8

1.6 exposure on the basis of metabolite levels [µg/kg b.w./day]

exposure on the basis of metabolite levels [µg/kg b.w./day]

Abbreviations: BMDL10, Benchmark Dose Lower Limit; MOE, margin of exposure. BMDL10 values from JECFA.20 astatistically significant difference between smokers and non-smokers (Po 0.00001). bsmoker’s excluded. cstatistically significant compared with other groups (Drip infusion; Acrylamide intake o10 μg/day) (Po0.05).

2.6 2.4 2.2

r = 0.26; p = 0.012

2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 -0.2 -0.1

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

exposure from the diet and tobacco smoke [µg/kg b.w./day]

1.4 r = 0.31; p = 0.004

1.2 1.0 0.8 0.6 0.4 0.2 0.0 -0.2 -0.1

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

exposure from the diet [µg/kg b.w./day]

Figure 1. Correlation between the acrylamide intake estimated on the basis of urinary levels of AAMA and GAMA and estimated on the basis of the 24-h dietary recall in the whole group of study subjects (n = 93).

Figure 2. Correlation between the acrylamide intake estimated on the basis of urinary levels of AAMA and GAMA and estimated on the basis of the 24-h dietary recall in the group of non-smokers (n = 88).

urinary levels of mercapturic derivatives of acrylamide and glycidamide, we performed the above assessment in the combined group of 88 women who were non-smokers (N = 78) and passive smokers (N = 10). On the basis of the 24-h dietary recall, we estimated the median dietary intake of acrylamide in the group of 88 women was 7.04 μg/day, ranging from 0 to 51.3 μg/day. The dietary intake of acrylamide estimated in the present study was almost three times lower than estimated in a probabilistic approach (20.43 μg/day) in our previous studies for the population of adult women (aged 19–96 years) in Poland.32 It was also approximately one half lower than the recently estimated,39 on the basis of the 24-h recall, lowest intake in a group of women in 10 European countries (range: 12–39 μg/day). Lower dietary intake of acrylamide in the current study is a result of the abovementioned type of diet (hospital diet) consumed by the study subjects. It should be emphasised that, similarly as in our previous study in 2010, the main source of acrylamide in the diet of the present study subjects was bread, followed by cookies, rusks and cakes. The results obtained support our previous conclusions that the main source of acrylamide in the diet of adults in Poland is bread and cereal products. Interestingly, similar results were obtained by Freisling et al.40 in the above-cited study conducted in 10 European countries, finding that bread, crispbread and rusks and in addition coffee accounted for at least 50% of the dietary intake of acrylamide.

In the whole group of 88 non-smoking women, AAMA and GAMA levels varied over a rather extensive range, from 2.3 to 148 μg/l and from 1.3 to 50.7, respectively, despite rather unvaried diet, similar with regard to the consumed products. It should be emphasised that in urine samples of women receiving only a drip infusion after delivery, we determined AAMA and GAMA at a level above LOQ, despite the fact that the study subjects did not intake acrylamide with the diet for at least 48 h. Our results confirm the results of Boettcher et al.,36 who found the presence of the above metabolites in the urine of a volunteer even after 46 h of fasting. The presence of mercapturic derivatives of acrylamide and glycidamide in the urine of women receiving only a drip infusion after delivery is difficult to explain. As shown by Tareke et al.41 in mice, after exposition to compounds which are known to generate hydroxyl radicals, for example, FeSO4 an increased internal dose of acrylamide occurred. They concluded the possibility of endogenous formation of acrylamide as a result of oxidative stress. In our study we determined acrylamide in drip solutions at the level below LOQ. For that reason we have assumed that the content of acrylamide in such low levels in drip solutions did not influence on its metabolite content in the urine. The presence of AAMA and GAMA in urine of women receiving only a drip infusion may indicate an unknown mechanism of endogenous formation of acrylamide. This requires further studies. Also in this group, as in the whole group of women participating in our study, a large variability in the levels of AAMA




7 and GAMA in urine was observed. This may be associated with different dynamics of acrylamide conversion to glycidamide and further to mercapturic derivatives, at a similar level of dietary intake. Our results seem to indicate the possibility of individually determined response to the exposure to acrylamide present in food. This may explain to a certain extent the difficulties in demonstrating in humans a direct correlation between the dietary intake of acrylamide and cancer development. On the other hand, it is worth noting that even a relatively small difference in acrylamide content in the diet after a period of physiological fasting (delivery) resulted in a small but significant (P o0.05) increase in the urinary level of AAMA (≥10 μg/day group vs o10 μg/day group). This seems to confirm the observation of Boettcher et al.36 that in humans acrylamide starts to be metabolised and eliminated as AAMA relatively rapidly after its oral administration. It is also interesting that, in contrast to AAMA, we did not find any significant differences in the median urinary level of GAMA between the study groups (drip infusion group vs o10 μg/day group vs ≥ 10 μg/day group). The median of estimated exposure to acrylamide in the whole group of 93 study subjects was 0.16 μg/kg b.w./day (1.15 μg/kg b.w./day P95) and was almost twice lower than estimated in a the probabilistic approach in our previous study for the entire population of adult women in Poland.32 The difference found resulted from an almost three times lower dietary intake of acrylamide estimated on the basis of the 24-h dietary recall in both the studies (7.04 μg/day vs 20.43 μg/day). The intake estimated by us was also six times lower than that estimated on the basis of the urinary level of AAMA in 31 children in Korea30 and 43 times lower than that estimated on the basis of the urinary levels of AAMA and GAMA in 110 children in Germany.29 It is generally known that acrylamide intake is higher in children due to the higher intake of food per kilogram body weight and higher intake of products that are the main source of acrylamide in the diet, including French fries and potato crisps. Exposure to acrylamide, in group of women within the first days after delivery, estimated on the basis of urinary levels of AAMA and GAMA, showed that even a slightly higher intake of acrylamide from diet resulted in a significant (P o0.05) increase in the exposure to this compound (0.22 μg/kg b.w./day vs 0.15 μg/kg b.w./day). Practically the same exposure was estimated by us on the basis on AAMA and GAMA concentration in urine and on the basis of the 24-h dietary recall (0.22 μg/kg b.w./day vs 0.23 μg/kg b.w./day) only in the group of women who consumed ≥ 10 μg of acrylamide daily. We found a weak but significant positive correlation between acrylamide intake calculated on the basis of the urinary levels of AAMA and GAMA and the intake estimated on the basis of 24-h dietary recall both for the entire study group of 93 women (r = 0.26, P o0.05) as well as for the group of 88 non-smoking women (r = 0.31, Po 0.005). It should be noted that, in a recent published study, Duarte-Salles et al.42 also found a weak positive correlation between the maternal acrylamide- and glycidamide-Hb adducts levels and the dietary acrylamide intakes estimated from the food frequency questionnaire during pregnancy. It seems that the weak association between urinary levels of AAMA and GAMA, similarly as levels of acrylamide- and glycidamide-Hb adducts and dietary acrylamide intake may result from the wide variation in the acrylamide content within the same food product group and even within the same product type. The estimated MOE values in present study in whole group of women ranged from 156 for 95th percentile to 1938 for median acrylamide intake. Significantly lower MOE values were found in smokers (range: 69–229), as well as in the group of women who consumed ≥ 10 μg of acrylamide daily (range: 305–1409). Despite of small group of smokers, our results seem to confirm that cigarette smoking may be associated with a high health risk. It should be emphasised that according to JECFA20 evaluation MOE values in the range from 45 to 310 may indicate a heath risk © 2015 Nature America, Inc.

associated with the acrylamide intake. Our results showed that even a small acrylamide level daily diet may be associated with health risk. This is particularly worrying due to the fact that they relate to women after childbirth. The acrylamide consumed in the diet by breastfeeding women can pass into breast milk and may pose a risk to the infant. In conclusion, we demonstrated for the first time in a relatively large group of women that mercapturic acids of acrylamide and glycidamide were detected in urine even after at least 48-h physiological fasting (after delivery). Regardless of the similar type of diet (hospital diet) we found a large variability in AAMA and GAMA concentration in urine of women participating in the study. This seems to indicate the possibility of individually determined response to exposure to acrylamide present in food. In addition, the presence of mercapturic derivatives of acrylamide and glycidamide in the urine of women receiving only a drip infusion after delivery may indicate the possibility of endogenous formation of acrylamide. The results obtained by us seem to confirm that cigarette smoking may be an important source of exposure to acrylamide. Our results also confirmed that the concentration of AAMA and GAMA in urine may be used for the assessment of dietary acrylamide exposure. As estimated by us, the dietary exposure of acrylamide to women after childbirth in most cases was low, with the exception of smokers. However, estimated MOE values were o10 000. This indicates that dietary intake of even relatively small amounts of acrylamide can be associated with health risks. CONFLICT OF INTEREST The authors declare no conflict of interest.

ACKNOWLEDGEMENTS This project was supported by a grant from the Ministry of Science and Higher Education (No. N N404 067740). We thank Professor Jürger Angerer and Dr Birgit Schindler for providing the standards of GAMA and GAMA-d3 to start the research. We also thank Dr Iwona Sajór and Ms Sylwia Gugała-Mirosz for conducting the 24-h dietary recalls and Ms Irena Stolarska for the excellent urinary creatinine level analyses.

REFERENCES 1 SNFA. Swedish National Food Administration. Information about acrylamide in food 2002: http://www.slv.se/engdefault.asp. 2 Mottram DS, Wedzicha BL, Dodson AT. Food chemistry: acrylamide is formed in the Maillard reaction. Nature 2002; 419: 448–449. 3 Stadler RH, Blank I, Varga N, Robert F, Hau J, Guy PA et al. Acrylamide from Maillard reaction products. Nature 2002; 419: 449–450. 4 Smith CJ, Perfetti TA, Rumple MA, Rodgman A, Doolittle DJ. “IARC Group 2A Carcinogens” reported in cigarette mainstream smoke. Food Chem Toxicol 2000; 38: 371–383. 5 Boettcher MI, Angerer J. Determination of the major mercapturic acids of acrylamide and glycidamide in human urine by LC-ESI-MS/MS. J Chromatogr B 2005; 824: 283–294. 6 Urban M, Kavvadias D, Riedel K, Scherer G, Tricker AR. Urinary mercapturic acids and a hemoglobin adduct for the dosimetry of acrylamide exposure in smokers and nonsmokers. Inhal Toxicol 2006; 18: 831–839. 7 He FS, Zhang SL, Wang HL, Li G, Zhang ZM, Li FL et al. Neurological and electroneuromyographic assessment of the adverse effects of acrylamide on occupationally exposed workers. Scand J Work Environ Heath 1989; 15: 125–129. 8 Hagmar L, Törnqvist M, Nordander C, Rosén I, Bruze M, Kautiainen A et al. Health effects of occupational exposure to acrylamide using hemoglobin adducts as biomarkers of internal dose. Scand J Work Environ Health 2001; 27: 219–226. 9 Johnson KA, Gorzinski SJ, Bodnar KM, Campbell RA, Wolf CH, Friedman MA et al. Chronic toxicity and oncogenicity study on acrylamide incorporated in the drinking water of Fisher 344 rats. Toxicol Appl Pharmacol 1986; 85: 154–168. 10 Friedman MA, Duak LH, Stedham MA. A lifetime oncogenicity study in rats with acrylamide. Fundam Appl Toxicol 1995; 27: 95–105.



8 11 Mucci LA, Dickman PW, Steineck G, Adami HO, Augustsson K. Dietary acrylamide and cancer of the large bowel, kidney and bladder: absence of an association in a population-based study in Sweden. Br J Cancer 2003; 88: 84–89. 12 Burley VJ, Greenwood DC, Hepworth SJ, Fraser LK, de Kok TM, van Breda SG et al. Dietary acrylamide intake and risk of breast cancer in the UK women’s cohort. Br J Cancer 2010; 103: 1749–1754. 13 Pelucchi C, La Vecchia C, Bosetti C, Boyle P, Boffetta P. Exposure to acrylamide and human cancer—a review and meta-analysis of epidemiologic studies. Ann Oncol 2011; 22: 1487–1499. 14 Olesen PT, Olsen A, Frandsen H, Frederiksen K, Overvad K, Tjønneland A. Acrylamide exposure and incidence of breast cancer among postmenopausal women in the Danish Diet, Cancer and Health study. Int J Cancer 2008; 122: 2094–2100. 15 Hogervorst JG, Schouten LJ, Konings EJ, Goldbohm RA, van den Brandt PA. Dietary acrylamide intake and the risk of renal cell, bladder, and prostate cancer. Amer J Clin Nutr 2008; 87: 1428–1438. 16 Hogervorst JG, Schouten LJ, Konings EJ, Goldbohm RA, van den Brandt PA. Lung cancer risk in relation to dietary acrylamide intake. J Natl Cancer Inst 2009; 101: 651–662. 17 Lin Y, Lagergren J, Lu Y. Dietary acrylamide intake and risk of esophageal cancer in a population-based case-control study in Sweden. Int J Cancer 2011; 128: 676–681. 18 International Agency for Research on Cancer (IARC). Acrylamide, IARC monographs on the evaluation of carcinogenic risks to humans. Some industrials chemicals. International Agency for Research on Cancer: Lyon, France, 1994; vol. 60: 389–433. 19 Bolger PM, Leblanc J – C, Setzer R.W. Application of the Margin of Exposure (MoE) approach to substances in food that are genotoxic and carcinogenic. EXAMPLE: Acrylamide (CAS No. 79-06-1). Food Chem Toxicol 2010; 48: S25–S33. 20 JECFA. Joint FAO/WHO Export Committee on food additives: evaluation of certain food additives and contaminants. 72nd report of the joint FAO/WHO expert committee on food additive. WHO Tech Rep Ser 2011; 959. http://whqlibdoc.who. int/trs/WHO_TRS_959_eng.pdf. 21 Fennell TR, Sumner SCJ, Snyder RW, Burgess J, Spicer R, Bridson WE et al. Metabolism and hemoglobin adduct formation of acrylamide in humans. Toxicol Sci 2005, 447–459. 22 Sumner SC, Fennell TR, Moore TA, Chanas B, Gonzalez F, Ghanayem BI. Role of cytochrome P450 2E1 in the metabolism of acrylamide and acrylonitrile in mice. Chem Res Toxicol 1999; 11: 1110–1116. 23 Bergmark E. Hemoglobin adducts of acrylamide and acrylonitrile in laboratory workers, smokers and nonsmokers. Chem Res Toxicol 1997; 10: 78–84. 24 Schettgen T, Weiss T, Drexler H, Angerer J. A first approach to estimate the internal exposure to acrylamide in smoking and non-smoking adults from Germany. Int J Hyg Environ Health 2003; 206: 9–14. 25 Boettcher MI, Schettgen T, Kütting B, Pischetsrieder M, Angerer J. Mercapturic acids of acrylamide and glycidamide as biomarkers of the internal exposure to acrylamide in the general population. Mutat Res 2005; 580: 167–176. 26 Von Tungeln LS, Doerge DR, Gamboa da Costa G, Marques MM, Witt WM, Koturbash I et al. Tumorigenicity of acrylamide and its metabolite glycidamide in the neonatal mouse bioassay. Int J Cancer 2012; 131: 2008–2015.


27 Sumner SCJ, Selvaraj L, Nauhaus SK, Fennell TR. Urinary metabolites from F344 rats and B6C3F1 mice coadministered acrylamide and acrylonitrile for 1 or 5 days. Chem Res Toxicol 1997; 10: 1152–1160. 28 Bjellaas T, Stølen LH, Haugen M, Paulsen JE, Alexander J, Lundanes E et al. Urinary acrylamide metabolites as biomarker for short-term dietary exposure to acrylamide. Food Chem Toxicol 2007; 45: 1020–1026. 29 Heudorf U, Hartmann E, Angerer J. Acrylamide in children – exposure assessment via urinary acrylamide metabolites as biomarkers. Int J Hyg Environ Health 2009; 212: 135–141. 30 Ji K, Kang S, Lee G, Lee S, Jo A, Kwak K et al. Urinary levels of N-acetyl-S(2-carbamoylethyl)-cysteine (AAMA), an acrylamide metabolite, in Korean children and their association with food consumption. Sci Total Environ 2013; 456-457: 17–23. 31 Brisson B, Ayotte P, Normandin L, Gaudreau É, Bienvenu J-F, Fennell TR et al. Relation between dietary acrylamide exposure and biomarkers of internal dose in Canadian teenagers. J Expo Sci Environ Epidemiol 2014; 24: 215–221. 32 Mojska H, Gielecińska I, Szponar L, Ołtarzewski M. Estimation of the dietary acrylamide exposure of the Polish population. Food Chem Toxicol 2010; 48: 2090–2096. 33 Szponar L, Wolnicka K, Rychlik E. Album fotografii produktów i potraw / Album of photographs of food products and dishes. Prace IŻŻ 96, 2000 Warszawa. 34 VITROS Chemistry Products CREA Slides – Instruction for use. http://apps. orthoclinical.com//TechDocs/TechDocSearch.aspx?culture = en-gb&tID = 0/ CREA_ J27323_EN_I.pdf. 35 Ciba-Geigy AG. Teiband Körperflüssigkeiten. In Leitner C (ed). Wissenschaftliche Tabellen Geigy. 8th edition. Ciba-Geigy AG: Basel, Switzerland, 1977; 51–97. 36 Boettcher MI, Bolt HM, Drexler H, Angerer J. Excretion of mercapturic acids of acrylamide and glycidamide in human urine after oral administration of deuterium-labelled acrylamide. Arch Toxicol 2006; 80: 55–61. 37 Mojska H, Gielecińska I, Stoś K. Zawartość akryloamidu w żywności w Polsce w świetle aktualnych zaleceń Unii Europejskiej / Acrylamide content in food in Poland in the light of current EU recommendations. Prob Hig Epidemiol 2011; 92: 625–628. 38 Mojska H, Gielecińska I, Świderska K. Zawartość akryloamidu w różnych rodzajach pieczywa w Polsce / Acrylamide content in various kind of bread in Poland. Bromat Chem Toksykol 2011; 64: 768–772. 39 Mojska H, Gielecińska I. Studies of acrylamide level in coffee and coffee substitutes: influence of raw material and manufacturing conditions. Rocz Panst Zakl Hig 2013; 64: 173–181. 40 Freisling H, Moskal A, Ferrari P, Nicolas G, Knaze V, Clavel-Chapelon F et al. Dietary acrylamide intake of adults in the European Prospective Investigation into Cancer and Nutrition differs greatly according to geographical region. Eur J Nutr 2013; 52: 1369–1380. 41 Tareke E, Lyn-Cook B, Robinson B, Ali SF. Acrylamide: a dietary carcinogen formed in vivo? J Agric Food Chem 2008; 56: 6020–6023. 42 Duarte-Salles T, von Stedingk H, Granum B, Gützkow KB, Rydberg P, Törnqvist M et al. Dietary acrylamide intake during pregnancy and fetal growth – results from the Norwegian Mother and Child Cohort Study (MoBa). Environ Health Perspect 2013; 121: 374–379.
