ToxSci Advance Access originally published online on September 6, 2007
Toxicological Sciences 2007 100(2):374-380; doi:10.1093/toxsci/kfm234
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Urinary Metabolites as Biomarkers of Acrylamide Exposure in Mice Following Dietary Crisp Bread Administration or Subcutaneous Injection
,1
* Norwegian Institute of Public Health, Division of Environmental Medicine, Nydalen, NO-0403 Oslo, Norway
Department of Chemistry, University of Oslo, Blindern, NO-0315 Oslo, Norway
Matforsk, Norwegian Food Research Institute, NO-1430 Aas, Norway
2 To whom correspondence should be addressed at Norwegian Institute of Public Health, Division of Environmental Medicine, PO Box 4404, Nydalen, NO-0403 Oslo, Norway. Fax: +47-22-04-22-43. E-mail: jan.erik.paulsen{at}fhi.no.
Received June 18, 2007; accepted August 31, 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Heat-treated carbohydrate-rich foods may contain high levels of acrylamide (AA). Crisp bread is a significant dietary AA source in the Nordic countries. We studied whether urinary metabolites of AA could be candidate biomarkers of AA intake and internal dose in mice following dietary crisp bread administration or sc injection. The crisp bread was experimentally baked to contain three different concentrations of AA: 0.19, 1.02, and 2.65 mg/kg, giving dietary exposures to AA of 0.024 ± 0.002, 0.14 ± 0.02, and 0.29 ± 0.04 mg/kg bodyweight (bw)/day (mean ± SD), respectively. A linear relationship was found between dietary AA exposure and urinary AA metabolites. On average, 55% of the ingested dose was recovered as urinary AA metabolites, and the molar proportions between the urinary metabolites showed similar proportions for the different doses. Urine AA metabolites were measured after sc injection of AA at doses of 0.05, 0.5, 5, and 50 mg/kg bw, and the urinary recovery for the three lowest doses was 54%. With the highest dose, 80% was recovered in urine, and the changed pattern of urinary metabolites indicated saturation of the metabolic conversion of AA to glycidamide. These results indicate that urinary metabolites of AA are good biomarkers of AA intake and internal dose up to 5 mg/kg bw/day. After sc injection of [14C]AA, 92% of the radioactivity was found in the urine and 2% in feces, liver, blood, and intestinal content (6% was not detected), demonstrating that sc AA was highly systemically available, that the major part AA metabolites was excreted, and that a significant portion of urinary AA metabolites (most likely glyceramide) was not accounted for by the present analytical method. Since the urinary recovery of AA after crisp bread feeding and sc injection was practically identical, an indicative "bioavailability" of AA from crisp bread was suggested to be approximately complete.
Key Words: urine; biomarkers; dietary exposure; acrylamide; glycidamide; food; bioavailability; crisp bread.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Significant amounts of acrylamide (AA) have recently been detected in a number of starch-rich foods consumed by the general human population. Maillard browning reaction products from mainly glucose and asparagine are responsible for the formation of AA (Mottram et al., 2002
; Stadler et al., 2002; Zyzak et al., 2003). Relatively high contents (up to 4000 µg/kg) of AA have been found in French fries (Tareke et al., 2002). The presence of AA in foods has caused considerable concern since AA is classified as probably carcinogenic to humans (2A) by the International Agency for Research on Cancer (IARC, 1994). The average daily intake of AA for adult humans, based on food consumption data from 17 different countries, has been estimated to be between 0.3 and 2.0 µg/kg bodyweight (bw) in adults and even higher in children (Dybing et al., 2005; JECFA, 2006).
A major urinary AA metabolite in both rats and mice is N-acetyl-S-(3-amino-3-oxopropyl)cysteine, a mercapturic acid AA (MA-AA) derivative (Sumner et al., 1992; Sumner et al., 1997). An alternative metabolic pathway is the CYP2E1-dependent oxidation of AA, from which the genotoxic epoxide glycidamide (GA) is formed (Sumner et al., 1999). GA reacts readily with DNA and other macromolecules (Dearfield et al., 1995; Segerback et al., 1995), and this is probably the main pathway responsible for the carcinogenic effect of AA. GA may be further metabolized by epoxide hydrolase to glyceramide (Kirman et al., 2003) or conjugated to glutathione. Following a stepwise conversion, it is excreted as the urinary mercapturic acid derivatives N-acetyl-S-(3-amino-2-hydroxy-3-oxopropyl)cysteine (MA-GA3) and N-acetyl-S-(carbamoyl-2-hydroxyethyl)cysteine (MA-GA2) (Sumner et al., 1992).
Recently, we developed a method to determine the mercapturic acids of AA and GA in human urine (Bjellaas et al., 2005). We used these as biomarkers for dietary AA intake in a clinical study and found immediate changes in the urinary AA-derived mercapturic acids following consumption of foods rich in AA (Bjellaas et al., 2006).
The objective of the present study was to (1) explore whether urinary metabolites of AA and GA could be candidate biomarkers of AA intake and internal dose in mice after dietary intake of AA from a regular food matrix by using an experimental crisp bread diet baked to contain different concentrations of AA and (2) compare the recovery of AA as urinary metabolites after dietary and sc exposure to provide indicative information about the fraction of AA from the crisp bread matrix that could be systemically available. Crisp bread is a significant AA source in the Nordic countries.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Chemicals and Stock Solutions
AA and N-isobutyryl-S-cysteine (NIBC) were obtained from Sigma-Aldrich (St Louis, MO). Deuterated AA [2,3,3-D3, 98%] was purchased from Larodan Fine Chemicals AG (Malmö, Sweden). GA (98%) was from Toronto Research Chemicals (North York, Ontario, Canada). Formic acid, hydrogen chloride (fuming, 37%), and potassium hydroxide (pellets) were from VWR (West Chester, PA). Methanol and acetonitrile were purchased from LabScan (Dublin, Ireland). The standards MA-AA, its deuterated analog (MA-AA[D3]), MA-GA2, MA-GA3, and N-acetyl-S-(allyl)cysteine were prepared as described elsewhere (Bjellaas et al., 2005
). Nitrogen gas was supplied by a Maxigas Nitrogen generator obtained from Dominick Hunter Ltd (Gateshead, UK) with purity of 99.999%. Type I water was supplied from a TKA GenPure water purification system (TKA Water Purification systems GmbH, Niederelbert, Germany). [14C]-AA was purchased from American Radiolabeled Chemicals Inc. (St Louis, MO). The chemicals for scintillation counting Ecosint A, Biosint, and Biosol were all purchased from Laborel (Uppsala, Sweden). Stock solution of GA (1 mg/ml) was made by dissolving GA in appropriate amount of type I water. Stock solution of AA[D3] (1 mg/ml) was made by dissolving AA[D3] in appropriate amount of type I water. An internal standard solution of AA[D3] (20 µg/ml) was prepared by diluting 400 µl of stock solution of AA[D3] to 20 ml with type I water.
Main calibration solution A was prepared by diluting 80 µl of stock solution to 20 ml type I water. Main calibration solution B was prepared by diluting 80 µl of stock solution to 4 ml type I water. Stock and calibration solutions for MA-AA, MA-GA2, and MA-GA3 were made according to Bjellaas et al. (2005).
Urine samples from mice not exposed to AA were used for preparing calibration samples. Calibration samples I and II were prepared by adding 25 and 50 µl of main calibration solution A to 1 ml of urine. Calibration samples III, IV, and V were prepared by adding 30, 50, and 75 µl of main calibration solution B to 1 ml of urine. To all calibration samples (I–V), 35 µl of concentrated formic acid and 30 µl of 20 µg/ml internal standard solution were added. All the calibration samples were diluted to 3.5 ml with type I water.
Animals and Diet
For the characterization of urinary excretion of AA metabolites from crisp bread feeding or sc injection of AA or GA, we used male C57BL/6JBomTac mice (Bomholt, Ry, Denmark), 7- to 8-weeks old. In a separate experiment, we studied the systemic availability, distribution, and excretion of sc administered [14C]AA using male mice of the same strain (C57BL/6J) from a different breeder (The Jackson Laboratory, Bar Harbor, ME), 17- to 19-weeks old. The mice were housed in plastic cages, three mice per cage, in a room with a 12-h light/dark cycle, controlled humidity (55 ± 5%), and temperature (20°C–24°C). All mice were housed at the Norwegian Institute of Public Health animal facility for at least a week before the study started.
Water and feed were given ad libitum. The mice were given a diet consisting of AIN-76 (Special Diet Services, Witham Essex, England) with 5% corn oil (control diet) or one of three different types of crunched crisp bread. These were produced from dough containing whole meal rye flour (53%), water (46%), and salt (0.9%) by baking for 15 min in an electrically heated stone oven (Bråthen and Knutsen, 2005). Type I of crisp bread, baked at 140°C, contained 191 µg AA/kg. Type II, baked at 160°C, contained 1020 µg AA/kg. Type III, baked at 210°C, contained 2650 µg AA/kg. The control diet was given to all mice not fed the crisp bread diet.
Protocol
Dietary exposure to AA.
To achieve steady state in AA dietary exposure, the mice were given crisp bread diets or control diet for 3 days while kept in plastic cages. Then, after moving the mice to metabolic cages, the 24-h pooled intake of diet and water from the three mice per cage was measured. In addition, the 24-h pooled urine from the same mice was collected in a tube constantly kept on ice. The 24-h recordings from groups of mice fed crisp bread of type I, II, or III (two cages per type) proceeded for 4 days (group sizes: n = 8). The 24-h recordings from groups of mice fed control diet (two cages) proceeded for 2 days (group size: n = 4).
Subcutaneous administration of AA or GA.
The mice were placed into metabolic cages (three mice per cage) and fed the control diet. After 1 day, the mice were injected sc with 0 (control), 0.05, 0.5, 5, or 50 mg/kg bw of AA, or GA, in 0.9% saline solution (10 µl/g Bw), two cages per treatment (n = 2). The 24-h pooled urine excreted in each metabolic cage was collected the subsequent 3 days, while the collecting tube was constantly kept on ice. The 24-h intake of diet and water was measured in the same periods.
Subcutaneous administration of [14C]AA.
Nine mice were placed into metabolic cages (three mice per cage) and fed the control diet. After 1 day, six mice (two cages) were injected sc with 10 µCi of [14C]AA/kg bw (0.16 mg/kg bw [14C]AA; 10 µl/g Bw), and three control mice (one cage) were injected with 0.9% saline solution. The 24-h pooled urine and feces in each metabolic cage was collected the subsequent 2 days, while the collecting tube was constantly kept on ice. The 24-h intake of diet and water was measured in the same periods. The mice were sacrificed with CO2 at 48 h after injection, and blood, liver, and intestinal content were collected.
Sample Preparation and Derivatization of Urinary GA
Sample preparation for the determination of the mercapturic acid metabolites of AA was performed by solid-phase extraction (SPE) as described previously by Bjellaas et al. (2005). For GA determination, the eluates from the sample application and washing step of the SPE were combined and used. GA was derivatized upon the addition of NIBC (56 µl of 1 mg/ml) to the combined sample forming N-isobuturyl-S-(3-amino-2-hydroxy-3-oxopropyl)cysteine (NIBC-GA3). Dissolved oxygen was removed from the solution by purging with N2 gas for 1 min. The solution was made basic by adding 250 µl 6M KOH and stirred magnetically (500 rpm) at 50°C for 30 min. The reaction mixture was acidified by adding 250 µl of 6M HCl prior to performing SPE using Oasis Hydrophilic-Lipophilic Balanced SPE columns (Waters, Milford, MA). The column was conditioned with 2 ml MeOH, 2 ml type I water, and 1 ml 1% Formic Acid (FA) in water prior to application of the acidified reaction solution. After washing with 1.5 ml 1% FA in water, NIBC-GA3 was eluted with 2 ml FA:MeOH:water (vol/vol, 1:80:19).
Urine samples from mice treated with [14C]AA were prepared for scintillation counting by adding 150 µl urine to 20 ml Ecosint A. Feces samples were prepared for scintillation counting by dissolving 50 mg of feces in 2 ml of Biosolv followed by incubation at 50°C for 3 h before 20 ml of Biosint were added. Liver samples were treated the same way as feces samples. Blood samples were centrifuged at 3000 rpm for 5 min. The resulting plasma was treated the same way as urine. The erythrocytes were added 2 ml of Biosolv and incubated at 50°C for 3 h. Twenty milliliters of Biosint was added to 500 µl of this incubated solution.
Determination of Urinary Metabolites
Urinary mercapturic acid metabolites from AA were determined by liquid chromatography with positive electrospray-ionization tandem-mass spectrometry (LC-MS/MS) as previously described by Bjellaas et al. (2005). Urinary GA (NIBC-GA3) was determined using the same LC-MS/MS instrumentation. Chromatography of NIBC-GA3 was achieved using a Hypersil Base Deactivated Silica column with dimensions 2.1 x 150 mm and 5 µm particles (ThermoHypersil-Keystone, Bellefonte, PA) using a flow rate of 250 µl/min. Solvents used as mobile phase were 0.1% formic acid in type I water (A) and acetonitrile (B). The mobile phase gradient was as follows: 0.0 min, 100% A; 1.0 min, 99% A; 2.0 min, 95% A; 3.0 min, 85% A; 5.0 min, 50% A; 6.0 min, 0% A; 8.0 min, 0% A; 8.1 min, 100% A; and 12.0 min, 100% A.
The NIBC-GA3 was detected by multiple reaction monitoring (MRM). The electrospray source settings were as follows: capillary voltage, 4 kV; cone voltage, 8 V; capillary temperature, 300°C; collision energy, 10 V; sheath gas pressure, 32 arbitrary units; and auxiliary gas pressure, 6 arbitrary units. Argon was used as the collision gas (1.5 mTorr). For all the MRM transitions, the dwell time was 0.2 s. Quantification was done by isotope dilution. The radioactivity in the samples was counted with a Packard Tri-Carb 1900CA liquid scintillation analyzer (Packard Instruments, Downers Grove, IL). The counting efficiency was controlled by a Packard automatic 14C-quenching standard.
Validation of GA Quantification
The method for quantifying the derivatized GA was validated using the calibration samples. Peak area ratios with respect to the internal standard were plotted against the corresponding concentration ratio. The response was found to be linear in the validated range (28.5–428.6 ng/ml urine) with a correlation coefficient of r2 > 0.98. Repeatability was tested on levels I, III, and V (Table 1) and was found to be 24, 20, and 5% RSD, respectively. The average accuracy as recovery, estimated at levels I, III, and V (Table 1), was found to be 100% at all three levels.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Molar Relationship between Urinary AA Metabolites and AA Exposure from Dietary Crisp Bread
Plotting all the 24-h records as molar intake of AA from crisp bread versus total molar excretion of urinary AA metabolites, it was found a good linear relationship between AA intake and excretion (Fig. 1A). No AA metabolites were detected in the urines collected from the mice fed the control diet. The mean dietary intakes of AA from the different types of crisp bread were calculated to be 24 ± 2 µg/kg bw/day, 143 ± 24 µg/kg bw/day, and 289 ± 35 µg/kg bw/day (± SD) for type I, II, and III diets, respectively. The mean fraction of the ingested dose recovered as urinary AA metabolites was 55 ± 8% (± SD) for the different crisp bread diets, and the molar proportions between the urinary metabolites showed a similar pattern for the different doses (Fig. 1B).
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Molar Relationship between Urinary AA Metabolites and AA Exposure from sc Injection
Again, a good linear relationship was found between the molar amount of AA administrated and the total molar amount of urinary AA metabolites excreted, even in the large dose range investigated (Fig. 2A). No AA metabolites were observed in the urine of the controls. For the three lowest doses injected, the mean fraction of the injected dose recovered as urinary AA metabolites was calculated to be 54 ± 3% (Fig. 2B). The recovery of the highest dose was significantly higher than that of the lower doses, and the metabolite pattern differed considerably from that of the lower doses with a much higher proportion of MA-AA relative to MA-GA3. This could be due to a saturation of CYP2E1-mediated conversion of AA to GA. The cumulative urinary excretion curves, expressed as recovery (%) of injected AA dose, shows that the major part was excreted during the first 24 h (Fig. 3).
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Fate and Recovery of [14C]AA from sc Injection
Since only about 54% of the lower doses of the AA administered sc was recovered in urine as mercapturic acid metabolites and GA, we investigated the fate of the remaining 45%. Therefore, mice were given a dose of radiolabeled [14C]AA sc. Urine, feces, liver, and blood samples were analyzed by liquid scintillation counting. The recovery of radioactivity in the urine was 92% at 48 h (Fig. 3). About 2% of the activity was recovered in feces, liver, intestinal contents, blood, and wash water of the cage (Table 2). The total radioactivity recovered from the administration of [14C]AA was calculated to be 94% (± 4 SD). The discrepancy between recovery in urine from sc [14C]AA administration and recovery in urine from sc AA administration strongly suggest a significant contribution of other urinary metabolites not determined by the methods used in the present study.
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Molar Relationship between Urinary GA Metabolites and GA Exposure from sc Injection
In order to examine the metabolism of GA and the amounts excreted as parent compound and as mercapturic acid metabolites, mice were given GA sc at doses 0, 0.05, 5, and 50 mg/kg bw. The major metabolite found in urine was MA-GA3. The recoveries of the determined urinary metabolites were dose dependent: 40, 55, and 66% for the doses 0.05, 5, and 50 mg/kg bw, respectively (Fig. 2D). As expected, the AA metabolite MA-AA was virtually absent in the urine of the GA-exposed animals. The minor amounts of MA-AA found in several urine samples can be attributed to an AA impurity in the commercial GA (98% purity).
A linear relationship was found between the amount of GA administered and the amount of urinary metabolites within the exposure range investigated (Fig. 2C). The recovery of only 40–66% of the given GA at increasing dose levels suggests the formation and urinary excretion of additional GA metabolites not accounted for by the method used in this study, possibly 2,3-dihydroxypropionamide (glyceramide) (previously determined by nuclear magnetic resonance, NMR; Sumner et al., 1992). Our LC-MS/MS method is not suitable for the determination of this highly polar compound, which could be responsible for the discrepancy in recoveries shown in Figure 3.
Comparison of Urinary Recoveries of AA Following Dietary and sc Route of Administration
Data from sc [14C]AA indicated that sc AA was virtually completely systemically available and that the major part was excreted in the urine (only 2% were detected elsewhere). Therefore, comparing the amount of AA recovered as urinary metabolites from crisp bread feeding (55%) with the recovered amount of AA as urinary metabolites from sc AA administration (54%) could provide some indicative information about the fraction of AA from the crisp bread matrix that might be systemically available. Since the recoveries of urinary metabolites from mice with dietary exposure to AA and mice sc administrated AA were found to be practically identical, this indicative "bioavailability" of AA from crisp bread appeared to be approximately complete.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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In the present study, we analyzed urinary metabolites of AA and GA in C57BL mice after crisp bread feeding and sc exposure to AA. The crisp bread was experimentally baked to contain three different concentrations of AA. There was a remarkable linear relationship between AA exposure and the total amount of urinary metabolites in the dose range 0.024–5 mg/kg bw/day independently of mode of administration. Furthermore, similar molar proportions between the urine metabolites were observed up to 5 mg/kg bw/day. Although the urinary metabolites detected only represent
55% of the dose administrated, they appear to be good biomarkers of AA intake and the internal dose. Oral bioavailability, which is defined as the fraction of the oral-administered dose that reaches the systemic circulation, is determined by comparing the fraction of parent compound and metabolites that accumulates in plasma over a given time with the similar fraction observed after iv administration. Even though the experimental design did not reveal the bioavailability of AA from crisp bread, the data provide indicative information that may be interpreted to frame a hypothesis about bioavailability. The main premiss for this hypothesis is data from sc [14C]AA, which indicated that sc AA was virtually completely systemically available and that the major part was excreted in the urine. Hence, this indicative bioavailability of AA from experimental crisp bread, baked to achieve different dose levels of AA, was found to be approximately 100% by comparing the amount AA recovered as urinary metabolites from steady-state feeding of the crisp bread diet (intake per day:urinary metabolites per day) with the amount recovered as urinary metabolites from a single dose of AA injected sc (dose injected:total amount metabolites excreted in 3 days).
There are only few data on bioavailability of AA from food in animal models (Doerge et al., 2005a,b). In mice (Doerge et al., 2005a), the bioavailability of AA was investigated by using a relatively low dose of AA, 0.1 mg/kg bw, given as gavage in water or as an AA-fortified diet and determined by comparing the sum of the normalized areas under the curves for AA in plasma for the oral administrations with that obtained following iv administration. The bioavailability was calculated to be about 23% from the fortified diet and 32–52% from the aqueous solution given by gavage. This low bioavailability compared with the indicative estimate in the present study could be due to the fact that they only measured the parent compound in plasma and that they did not take into the account the first pass metabolism of AA to GA. The toxicokinetic situation could be even more complex since they determined the bioavailability of GA given by gavage to be 120%. Another difference between the studies was the AA feeding procedure: (Doerge et al., 2005a) trained the mice to consume a single meal within a 30-min time interval while we feed the mice continuously for several days to achieve a steady-state condition, which included the coprophagous behavior (the rodent eating off soft feces with cecum content). These results indicate that a broad spectrum of plasma metabolites of AA are needed to be measured for the determination of the bioavailability.
The discrepancy between recovery in urine from sc [14C]AA administration (92%) and recovery in urine from sc AA administration (55%) strongly suggest a significant contribution of urinary metabolites not determined by the methods used in the present study. Two observations indicate that urinary glyceramide could be the main candidate: the recovery of the highest AA dose was significantly higher than that of the lower doses with a simultaneous reduction in the conversion of AA to GA (higher proportion of MA-AA relative to MA-GA3) and the recovery of GA following administration as urinary MA-GA2, MA-GA3, and GA was far from 100%. It has previously been shown that AA oxidation in vivo was greater at lower doses than at higher doses (Bergmark et al., 1991). The suggested reduced conversion of AA to GA at the highest AA dose (50 mg/kg bw) could be due to saturation of CYP2E1 metabolic capacity. Likewise, the relative increase in the amount excreted as mercapturic acid metabolites with increased GA dose could indicate saturation of the hydrolytic pathway (epoxy hydrolase) to glyceramide. In our study, it appears that a significant fraction of the total amount of AA converted to GA is further converted to the GA metabolite glyceramide. Unfortunately, we had no method available for determination of this highly polar compound.
Fennell and Friedman (2005) dosed humans with 3 mg/kg bw of 13C-AA and used NMR analysis to investigate urinary metabolites. They found that in humans, the major urinary metabolite derived from GA was glyceramide (about 11% of the total urinary metabolites found). Hence, to assess bioavailability of dietary AA in humans, it would in addition to the mercapturic acid metabolites also be necessary to quantify glyceramide in human urine. Development of such a method should be a priority.
Mice given crisp bread showed a lower conversion to GA compared with that we observed following sc administration of AA at 5 mg/kg bw and below. We do not know the reason for this difference, but it could be due to unknown toxicokinetic differences related to long-term steady-state feeding of crisp bread versus single sc injection.
For the risk assessment of AA, it is important to determine the conversion rate of AA from food to the genotoxic epoxide GA as well as the rate of GA removal and to compare the rates in humans to those in rodents, since current cancer risk extrapolation is based on rodent studies. By injecting GA, it was possible to investigate the fate of systemically available GA as urinary mercapturic acid metabolites. The linear relationship between systemic GA and the total amount of these metabolites indicates that they may function as biomarkers of internal GA dose in the present model. However, for comparing the internal GA dose between species, which may vary in enzyme characteristics, it seems necessary to determine all the urinary metabolites, including glyceramide.
In conclusion, the urinary GA and mercapturic acid metabolites of AA and GA, which represent 55% of the AA intake in mice, proved to be good biomarkers for AA intake and internal dose. The indicative bioavailability of AA from crisp bread was close to 100%.
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FUNDING |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Nordic Innovation Center (Contract 04005, "NORDACRYL: AA precursors, limiting substrates, and in vivo effects"), the Research Council of Norway, Nordic Council of Ministers and the European Commission, Priority 5 on Food Quality and Safety (Contract No. FOOD-CT-2003-506820 Specific Targeted Project), "Heat-generated food toxicants—identification, characterization, and risk minimization."
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NOTES |
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1 These authors contributed equally to this study.
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ACKNOWLEDGMENTS |
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We thank Hege Hjertholm, Tone Rasmussen, André Larsen, and Victor Labay Ong for great help with the animals. The AA analysis of the crisp breads has been performed by Finnish Food Safety Authority-Evira under the NORDACRYL project. This publication reflects the author's views and not necessarily those of the European Commission. The information in this document is provided as is, and no guarantee or warranty is given that the information is fit for any particular purpose. The user thereof uses the information at its sole risk and liability.
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