Validation of putative biomarkers of furan exposure through quantitative analysis of furan metabolites in urine of F344 rats exposed to stable isotope labeled furan

Humans are chronically exposed to furan, a potent liver toxicant and carcinogen that occurs in a variety of heat-processed foods. Assessment of human exposure based on the furan content in foods is, however, subject to some uncertainty due to the high volatility of furan. Biomarker monitoring is thus considered an alternative or complementary approach to furan exposure assessment. Previous work suggested that urinary furan metabolites derived from the reaction of cis-2-butene-1,4-dial (BDA), the reactive intermediate of furan, with glutathione (GSH) or amino acids may serve as potential biomarkers of furan exposure. However, some metabolites were also reported to occur in urine of untreated animals, indicating either background contamination via animal feed or endogenous sources, which may limit their suitability as biomarkers of exposure. The overall aim of the present study was to accurately establish the correlation between external dose and concentration of furan metabolites in urine over time and to discriminate against endogenous formation and furan intake via feed. To this end, the furan metabolites GSH-BDA (N-[4-carboxy-4-(3-mercapto-1H-pyrrol-1-yl)-1-oxobutyl]-L-cysteinylglycine), NAcLys-BDA (R-2-(acetylamino)-6-(2,5-dihydro-2-oxo-1H-pyrrol-1-yl)-1-hexanoic acid), NAcCys-BDA-NAcLys (N-acetyl-S-[1-[5-(acetylamino)-5-carboxypentyl]-1H-pyrrol-3-yl]-L-cysteine) and NAcCys-BDA-NAcLys sulfoxide (N-acetyl-S-[1-[5-(acetylamino)-5-carboxypentyl]-1H-pyrrol-3-yl]-L-cysteine sulfoxide) were simultaneously analyzed by stable isotope dilution ESI–LC–MS/MS as unlabeled and [13C4]-furan dependent metabolites following oral administration of a single oral dose of isotopically labelled [13C4]-furan (0.1, 1, 10, 100 and 1000 µg/kg bw) to male and female F344/DuCrl rats. Although a linear correlation between urinary excretion of [13C4]-furan-dependent metabolites was observed, analysis of unlabeled NAcLys-BDA, NAcCys-BDA-NAcLys and NAcCys-BDA-NAcLys sulfoxide revealed substantial, fairly constant urinary background levels throughout the course of the study. Analysis of furan in animal feed excluded feed as a source for these background levels. GSH-BDA was identified as the only furan metabolite without background occurrence, suggesting that it may present a specific biomarker to monitor external furan exposure. Studies in humans are now needed to establish if analysis of urinary GSH-BDA may provide reliable exposure estimates. Supplementary Information The online version contains supplementary material available at 10.1007/s00204-024-03722-5.


Introduction
The process related food contaminant furan is formed in a range of heat-treated food items due to thermal degradation of natural food components.Relatively high levels of furan are found in coffee, coffee products and canned food, particularly processed baby food.Based on the concentration of furan in food and dietary surveys across European countries, highest dietary exposures to furan were estimated in infants and adults (European Food Safety Authority).In infants, mean and 95th percentile exposures ranged between 0.14 and 0.99 µg/kg bodyweight (bw) per day (minimum lower bound (LB) to maximum upper bound (UB)) and 0.19 to 1.82 µg/kg bw per day (minimum LB to maximum UB), respectively.Chronic dietary exposure in adults was estimated to range between 0.11 and 0.75 µg/kg bw per day (minimum LB to maximum UB) on average and between 0.20 and 1.22 µg/kg bw per day (minimum LB to maximum UB) in highly exposed consumers (95th percentile) (European Food Safety Authority 2017).
Based on the margin of exposure approach (MOE), EFSA concluded in its 2017 risk assessment that the current exposures to furan, a potent hepatotoxin and hepatocarcinogen, indicate a health concern (European Food Safety Authority 2017).For non-neoplastic effects, the MOEs calculated using a Benchmark dose lower confidence limit 10% (BMDL 10 ) of 0.064 mg/kg bw per day for induction of cholangiofibrosis in male rats after 2 years as the point of departure were below 100 for some age groups, such as infants and toddlers, and were therefore considered to indicate a health concern.For neoplastic effects, EFSA selected a BMDL 10 of 1.31 mg/kg bw per day for increased incidence of hepatocellular adenomas and carcinomas in female mice after 2 years as reference point.The calculated MOEs for neoplastic effects were smaller than 10,000 for most dietary surveys.However, EFSA emphasized the uncertainties in the present exposure assessment, which may lead to both underand overestimation of dietary furan exposure (European Food Safety Authority 2017).In particular, furan formation during home cooking as well as evaporation losses of furan during standing of beverages after brewing (e.g., coffee) or reheating of commercially processed foods (e.g., ready-toeat meals for infants) were not considered in the assessment (European Food Safety Authority 2017).
In view of the background levels and limited quantitative data on furan metabolite excretion following oral intake, the aim of the present study was to further assess furan metabolites as potential biomarkers of furan exposure and to discriminate between the externally applied dose and background exposure through quantitative analysis of furan-dependent metabolites in urine of rats after a single oral dose of isotopically labeled furan.To this end, GSH-BDA, NAcLys-BDA, NAcCys-BDA-NAcLys and its corresponding sulfoxide were simultaneously quantified by stable isotope dilution ESI-LC-MS/ MS as unlabeled and [ 13 C 4 ]-furan dependent metabolites in urine of male and female F344/DuCrl rats administered [ 13 C 4 ]-furan by oral gavage.To establish a correlation between external dose and urinary excretion of furanderived metabolites, furan was administered across a wide dose range that includes doses relevant to human exposure (0, 0.1, 1, 10, 100 and 1000 µg/kg bw).
Fig. 1 Metabolic pathways of furan and potential urinary biomarkers of furan exposure.Furan is predominantly metabolized by CYP 2E1, leading to the formation of the reactive dialdehyde cis-2-butene-1,4-dial (BDA).The reactivity of BDA towards cellular nucleophiles results in a broad spectrum of urinary metabolites: GSH-BDA (1), the product of the conjugation with glutathione and a subsequent intramolecular reaction; NAcLys-BDA (2), the adduct of BDA and lysine, followed by acetylation of the ⍺-amino group of lysine; NAc-Cys-BDA-NAcLys (3) and its corresponding sulfoxide (4), which derive from crosslinks of cysteine and lysine by BDA and subsequent N-acetylation and oxidation.Note that conjugation with GSH and cysteine can also occur in position 2 instead of 3 in the pyrrol ring

Chemical syntheses
Reference compounds were prepared by reacting BDA with GSH or amino acid derivatives as previously described (Chen et al. 1997;Lu et al. 2009;Hamberger et al. 2010;Karlstetter and Mally 2020).

Animal experiments
Animal experiments were performed according to national animal welfare regulations after authorization by the local authorities (Regierung von Unterfranken, AZ RUF-55.2.2-2532-2-1256-20).A total of sixty 6-7 weeks old male and female F344/DuCrl rats (Charles River, Sulzfeld, Germany) were housed in groups of five in Macrolon cages with free access to sterilized (25 kGy) pelleted standard rat maintenance diet (SSNIFF, Soest, Germany) and tap water to acclimatize for 12 days prior to treatment.Room temperature was maintained at 22 ± 2 °C with a relative humidity of 55 ± 10% and a day/night cycle of 14/10 h.Rats were transferred into individual metabolic cages 24 h prior to treatment with free access to sterilized (25 kGy) ground standard rat maintenance and tap water ad libitum.Rats (n = 5 per dose and sex, bw males: 142-180 g; bw females: 110-129 g) received a single dose of [ 13 C 4 ]-furan dissolved in corn oil (4 mL/kg bw) at doses of 0, 0.1, 1, 10, 100 and 1000 µg/kg bw by oral gavage.
Dosing solutions were freshly prepared immediately prior to administration.The entire content of a glass ampulla containing 10 mg (nominal amount) [ 13 C 4 ]-furan (cooled to −80 °C to prevent evaporation loss) was first diluted in 10 mL corn oil.This solution was further diluted with corn oil to prepare stock solutions containing 50 and 450 µg/mL [ 13 C 4 ]-furan.These stock solutions served to prepare 0.25 and 0.025 µg/mL dosing solutions for the 0.1 and 1 µg/kg bw dose groups, and 2.5, 25 and 250 µg/mL dosing solutions for the 10, 100 and 1000 µg/kg bw dose groups, respectively.Considering the volatility of furan, an aliquot of each stock solution (50 and 450 µg/mL) was retained for subsequent GC-MS analysis to confirm the [ 13 C 4 ]-furan content (2.6.HS-GC-MS analysis of [ 13 C 4 ]-furan stock solutions).
Urine samples were collected on ice for 24 h prior and for 6 days after treatment.During the first 24 h after treatment, urine was collected every 8 h, followed by collection intervals of 24 h until sacrifice.Urine volume was recorded at the end of the collection period and aliquots were stored at −80 °C until further analysis.To reduce furan intake via animal feed during acclimatization and throughout the study, sterilized (25 kGy) pelleted and ground feed was used instead of autoclaved feed.The furan content in animal feed was determined by HS-GC-MS as described below (2.7).

Sample preparation
Urine aliquots were thawed at 4-8 °C and vortexed.Urine was diluted with water (LC-MS/MS grade) at dilution factors of 2-200 to achieve analyte concentrations within the linear range of the calibration curves.To a volume of 90 µL diluted urine, 10 µL of internal standard mix and 1 µL of 8 M HCl were added.The internal standard mix contained isotopically labelled

LC-MS/MS analyses
LC-MS/MS analyses were performed with a Triple Quad 5500+ QTRAP mass spectrometer (Applied Biosystems/ MDS Sciex, Darmstadt, Germany) coupled to an Agilent 1100 HPLC and Agilent 1100 autosampler (Agilent, Waldbronn, Germany).Analytes (10 µL per sample) were separated on a Synergi Polar-RP analytical column (4 µm, 150 × 2 mm, 80 Å, Phenomenex Inc.) with water (containing 0.1% (v/v) formic acid) as solvent A and methanol (containing 0.1% (v/v) formic acid) as solvent B at a flow rate of 0.3 mL/min.Solvent A was held at 100% for 3 min, followed by a linear gradient to 20% A/80% B in 7 min.These conditions were held for 2 min before decreasing to 10% A/90% B. After 2 min at 90% B, the gradient was returned to initial conditions of 100% A/0% B within 2 min and remained until the end of the run (22 min).
Mass spectrometric analysis was performed using electrospray ionization operating in positive ion mode and multiple reaction monitoring (MRM) mode with an ion spray voltage of 5500 V and a source temperature of 500 °C.Nitrogen was used as ion spray (50 psi), drying gas (60 psi), curtain gas (40 psi) and collision gas.Compound specific ESI-MS/ MS-parameters are given in Table 2. Data were recorded by Analyst 1.7.3 software (Applied Biosystems/MDS Sciex, Darmstadt, Germany).
Seven-point calibration curves were prepared by spiking water with appropriate volumes of working standard solution, containing 100 ng/mL of each metabolite.Calibration was linear in the range of 1.25-80 ng/mL.Working standard solution was prepared from individual stock solutions (1 mg/ mL) of the respective metabolite in water.Due to the low yield of GSH-[ 13 C 4 ]-BDA and limited availability/significant costs of 2,5-diacetoxy-2,5-[ 13 C 4 ]-dihydrofuran required for synthesis, quantitation of GSH-[ 13 C 4 ]-BDA was performed using GSH-BDA.To 90 µL of each calibration standard 10 µL of internal standard mix and 1 µL of 8 M HCl were added.Solutions were then vortexed and centrifuged at 4 °C and 15,000 g for 10 min.The peak area ratios (analyte peak area/internal standard peak area) were used for quantitation.To ensure method precision during the analytical run, quality controls of 20 ng/mL were measured after every tenth sample.Limits of detection (LOD) and quantitation (LOQ) for each reference substance defined as a signal-to-noise ratio of 1:3 and 1:7, respectively, are provided in Table 3.

HS-GC-MS analysis of [ 13 C 4 ]-furan in stock solutions
The [ 13 C 4 ]-furan content of the stock solutions used to prepare the dosing solutions was analyzed by headspace GC-MS analysis following the FDA standard addition method for determination of furan in foods with minor modifications (Food and Drug Administration 2004).Briefly, GC-MS analysis was carried out using an Agilent 6890 GC coupled to an Agilent 5973 MSD (Hewlett-Packard).
Chromatographic separation was performed on a HP-Plot Q capillary column (30 m × 0.32 mm, 20 µm phase thickness) with helium as carrier gas at a constant flow rate of 1.0 mL/min.The gas chromatograph operated with a split ratio of 10:1 and an injector temperature maintained at 200 °C.The gas chromatographic oven program started with an initial temperature of 50 °C for 1 min, an increase to 260 °C at a rate of 20 °C/min.The temperature was then held at 260 °C for 2.5 min.The total chromatographic run time was 14 min.The mass spectrometer was operated in electron-ionization mode at 70 eV with a source temperature of 230 °C and a MS-quad temperature of 150 °C.Data were acquired in selected ion monitoring (SIM) mode.The following ions were selected as quantifier (Qn) and qualifier (Ql): [ 13 C 4 ]-furan, m/z 72 (Qn) and 42 (Ql); furan, m/z 68 (Qn) and 39 (Ql); acetone, m/z 43 (Qn) and 58 (Ql).Dwell time was set at 50 ms for each ion.The retention time of [ 13 C 4 ]-furan and furan was 8.45 min, while acetone used as internal standard eluted at 9.05 min.The concentration of [ 13 C 4 ]-furan in the stock solutions was determined using standard addition.To obtain standard addition curves, stock solutions containing a nominal concentration of 50 µg/mL [ 13 C 4 ]-furan in corn oil were spiked with 25, 50 and 100 µg furan using a working solution of 5 mg/mL furan in methanol.Stock solutions of 450 µg/mL [ 13 C 4 ]-furan in corn oil were spiked with 250, 500 and 1000 µg furan using a working solution of 50 mg/mL furan in methanol.Working solutions were freshly prepared before use.Samples were fortified with 20 µL of internal standard solution of acetone in methanol, using a 50 mg/mL internal standard solution for 450 µg/mL stock solutions and a 5 mg/mL internal standard solution for 50 µg/mL stock solutions.Following incubation for 10 min at 60 °C, a volume of 1 mL gas phase was injected into the GC-MS system.Quantitation was conducted by evaluating peak areas in TIC modus and by plotting the area ratio of analyte and internal standard against the amount of spiked furan.

HS-GC-MS analyses of furan in animal feed
Furan was analyzed using a headspace-gas chromatography/ mass spectrometry (HS-GC-MS) procedure, initially developed and validated for baby foods (Lachenmeier et al. 2009).
Briefly, approximately 1 g of animal feed sample was cryomilled and added to a headspace vial with 90 mL water and measured using HS-GC-MS.Deuterated furan (furan-d 4 ) was used as internal standard, and the quantification was conducted using a multipoint calibration method.Analysis was conducted on a gas chromatograph coupled with mass spectrometer (Agilent 6890/5973) and a headspace sampler Combi PAL MXY 02-01B (Agilent, Waldbronn, Germany).Gas chromatography column: HP-Plot Q, 30 m, 0.32 mm I.D., film 20 µm (Agilent, Waldbronn, Germany).For further methodological details, see (Lachenmeier et al. 2009).

Results
To discriminate between the externally applied furan dose and background exposure via feed and/or endogenous formation and to establish the correlation between external dose and biomarker concentration, urinary excretion of concentrations than the nominal concentrations (Supplementary Table S1), presumably due to a slightly higher than nominal amount of [ 13 C 4 ]-furan supplied in the glass ampulla.Based on these results, the administered doses were corrected accordingly (nominal dose: 0.  6,16,199,1988,19,880 nmol/kg bw) (Supplementary Table S1).Excretion rates of [ 13 C 4 ]-furan metabolites were calculated based on the actual dose applied.
Based on the 24 h-exretion rates established via analysis of [ 13 C 4 ]-furan-dependent metabolites (Table 5), excretion of unlabeled NAcCys-BDA-NAcLys and NAcCys-BDA-NAcLys sulfoxide was estimated to correspond to a furan dose of 46 and 70 µg/kg bw/d in males and 94 and 135 µg/ kg bw/d in females.To understand if the presence of furan in animal feed may account for these background levels, the content of furan in 25 kGy sterilized animal feed used in the present study vs. standard autoclaved animal feed was assessed by HS-GC-MS.In contrast to autoclaved feed, Fig. 3 Urinary excretion of GSH-BDA, NAcLys-BDA, NAc-Cys-BDA-NAcLys and NAcCys-BDA-NAcLys sulfoxide as [ 13 C 4 ]-furan-derived metabolites (filled symbols) and corresponding unlabeled compounds (open symbols) in urine of male (A) and female (B) F344/DuCrl rats.Inserts present zoomed plots of the highlighted areas of the diagrams, showing metabolite excretion in the lower dose groups (100-1 µg/kg bw [ 13 C 4 ]-furan).Data are presented as mean ± standard deviation (n = 5) of the amount of metabolite excreted within 8 h ◂ Table 4 Urinary 24 h excretion of GSH-[ 13 C 4 ]-BDA, NAcLys-[ 13 C 4 ]-BDA, NAcCys-[ 13 C 4 ]-BDA-NAcLys, NAcCys-[ 13 C 4 ]-BDA-NAcLys sulfoxide expressed as µg/24 h, nmol/24 h and fraction of the administered dose (%) a after treatment of male and female F344/DuCrl rats with a single dose of [ 13 C 4 ]-furan, as well as 24 h background excretion of NAcLys-BDA, NAcCys-BDA-NAcLys and NAcCys-BDA-NAcLys sulfoxide a Dose adjusted based on analysis of the [ 13 C 4 ]-furan concentration in the stock solutions b Actual doses for the 10, 100 and 1000 µg/kg bw dose groups reported in the table were rounded to two significant digits (actual dose males: 14, 141, 1410 µg/kg bw; actual dose females: 14, 143, 1430 µg/kg bw) which was found to contain furan at 35 µg/kg feed, furan was below the LOQ (< 5 µg/kg) in 25 kGy sterilized ground animal feed.Pelleted 25 kGy sterilized animal feed that was fed during acclimatization up to 24 h before the start of the study was found to contain furan at 7.5 µg/kg feed.Based on the LOQ of 5 µg furan/kg feed and feed consumption data (mean daily feed consumption: 16.5 ± 3.3 g/d), furan exposure of animals via feed was estimated at < 0.6 µg/kg bw per day.Estimated intake of furan via feed (0.6 µg/kg bw) was thus about two orders of magnitude lower than background exposure estimated based on biomarker excretion (Table 5).In particular, the high background levels of NAcLys-BDA, which by far exceeded the levels of the BDA derived lysine-cysteine crosslinks, were estimated to correspond to a theoretical furan dose of 1.6 mg/kg bw per day in males and 2.1 mg/kg bw per day in females.

Discussion
The overall aim of the present study was to test the validity of a biomarker-based approach to assess exposure to furan via food.Administration of isotopically labelled [ 13 C 4 ]-furan to rats across a wide dose range that included doses relevant to human exposure (0.1-10 µg/kg bw) allowed us to precisely establish the correlation between external dose and  concentration of furan metabolites in urine over time and to discriminate against endogenous formation and furan intake via feed.Although a linear cor relation was observed between [ 13 C 4 ]-furan dose and urinary excretion of [ 13 C 4 ]-furan-dependent metabolites, the high background levels of NAcLys-BDA, NAcCys-BDA-NAcLys and NAcCys-BDA-NAcLys sulfoxide render these metabolites unsuitable as biomarkers of dietary furan.In particular, the constant high urinary background levels of NAcLys-BDA in the range of NAcLys-[ 13 C 4 ]-BDA concentrations observed in response to the highest dose of [ 13 C 4 ]-furan (1 mg/kg bw) and estimated to correspond to a furan intake of 1.6 and 2.1 mg/kg bw in male and female rats, respectively, provide clear evidence for a significant but yet unexplored source of furan or its metabolites.While our analyses of furan in animal feed appear to exclude animal feed as a significant source of furan exposure under the experimental conditions of our study, we cannot rule out the possibility that amino acid adducts of BDA, such as Lys-BDA, may be present in feed.This may be supported by the observation that urinary concentrations of NAcLys-BDA first declined during the day-time collection period on day 1 (8 h) and then continuously increased at the two later urine collection periods (16 and 24 h), and, therefore, corresponded with the animals´ nocturnal activity and eating/drinking behaviour.Alternatively, there is some evidence to suggest that furan and 2-butene-1,4-dial may be formed endogenously.Suggested pathways for endogenous formation include lipid peroxidation and 5'-oxidation of deoxyribose.Similar to the reactions that occur in food, oxidation of endogenous polyunsaturated fatty acids has been proposed to generate furan via formation and cyclocondensation of 4-hydroxy-2-butenal (Onyango 2012).In contrast to 4-hydroxy-2-nonenal (HNE), a well-established endogenous lipid peroxidation product, endogenous generation of other 4-hydroxy-trans-2-alkenals such as 4-hydroxy-2-butenal has, however, not been demonstrated (Rietjens et al. 2022).In addition to lipid peroxidation, formation of trans-2-butene-1,4-dial by 5'-oxidation of deoxyribose has been suggested as a possible endogenous source of BDA-amino acid adducts (Chen et al. 2004), but experimental proof that this occurs at significant rates in mammalian cells and gives rise to the same type of adducts with GSH or lysine as cis-2-butene-1,4-dial (BDA) is still lacking.However, it is conceivable that trans-2-butene-1,4dial formed from 5'-oxidation of deoxyribose may immediately react with close-by lysine residues, such as lysine residues on histones, which may be subsequently degraded to release Lys-BDA.The hypothesis that lysine residues on histones may be targeted by 2-butene-1,4-dial and its primary GSH-adduct is supported by identification of a cross-link between the GSH-BDA conjugate and lysine 107 of histone H2B in livers of rats treated with furan (Nunes et al. 2016).Based on the available data, it is presently not possible to conclude on the origin of the high background of BDA-derived lysine adducts and lysine-cysteine cross-links.As emphasized in a recent review, however, understanding the role of endogenous versus exogenous sources of process related food contaminants is critical for comprehensive exposure and risk assessment (Rietjens et al. 2022).In the case of furan-derived metabolites, it is evident that-if not considered in human biomonitoring studies-the high background levels of potentially endogenously formed metabolites excreted via urine may lead to an overestimation of furan exposure via food and consequently to an overestimation of the related human risk.
In contrast to NAcLys-BDA, NAcCys-BDA-NAcLys and NAcCys-BDA-NAcLys sulfoxide, which are thought to arise primarily from the reaction of BDA with proteinbound lysine and cysteine residues, GSH-BDA showed no background excretion.The absence of background levels and close correlation between external dose and 24 h excretion of GSH-[ 13 C 4 ]-BDA support GSH-BDA as a specific biomarker to monitor external exposure to furan.Excretion rates of GSH-[ 13 C 4 ]-BDA were low (< 2.5% within 24 h).This is, however, consistent with previous work demonstrating elimination of 20% of the orally ingested furan dose via urine in form of < 10 different metabolites (Burka et al. 1991), which arise from alkylation and cross-linking of GSH and free and protein-bound amino acids by the bifunctional electrophile BDA.The low excretion rates of GSH-BDA may prove difficult for human biomonitoring, in particular for translating human urinary biomarker concentrations into probable daily intakes, as minor differences in excretion rates such as those observed between male and female rats may have a significant impact on calculated intakes.Thus, studies in humans are now needed to test if biomonitoring of GSH-BDA is able to provide reliable exposure estimates.To this end, human toxicokinetic studies on furan similar to the present study in rats may be valuable to accurately determine GSH-BDA excretion rates in humans, also considering potential gender differences as suggested by our rat data.
In a recent study, elimination kinetics of the furan metabolites GSH-BDA, Lys-BDA and NAcLys-BDA and corresponding metabolites of 2-methylfuran were assessed in human volunteers after consumption of 500 mL of coffee brew containing a defined amount of furan and 2-methylfuran (Kremer et al. 2023).Participants were reported to eliminate 89.1 ± 21% of the ingested furan dose in urine within 24 h, with Lys-BDA accounting for 10.6 ± 4.4%, NAcLys-BDA accounting for 78 ± 18%, and consequently GSH-BDA accounting for less than 1% of the ingested dose (Kremer et al. 2023).While the low excretion rates of GSH-BDA are consistent with data in rats, the conclusion that in humans almost 90% of the furan dose are eliminated in urine is at odds with the present and previous studies in rats and extensive binding of furan to tissue proteins (Burka et al. 1991;Karlstetter and Mally 2020).Speciesdifferences in renal vs. biliary excretion may contribute to this discrepancy.A possible alternative explanation is that background levels of NAcLys-BDA and Lys-BDA appear not to have been taken into account in the human study.This may lead to overestimation of excretion rates and consequently wrong estimates of human exposure when translating biomarker data into probable daily intakes.The overall conclusion that Lys-BDA and NAcLys-BDA may be suitable as short-term biomarkers of furan exposure (Kremer et al. 2023) is not supported by our present data.Interestingly, overall excretion rates of 2-methylfuranderived metabolites were reported to be significantly lower (15.4 ± 4.8%) as compared to furan metabolites (89.1 ± 21%) (Kremer et al. 2023).Lys-AcA and NAcLys-AcA, i.e., two metabolites derived from acetyl acrolein, the reactive intermediate formed by cytochrome P450 dependent oxidation of 2-methylfuran, were also detected in human volunteers prior to coffee consumption.Whether this is due to intake of 2-methylfuran via food or potential endogenous formation is currently unclear.Although a pathway for formation of 2-methylfuran from omega-3 fatty acids via 2-hydroxy-2-pentenal has been proposed (Onyango 2012), there appears to be no evidence for potential endogenous formation of 2-methylfuran or its reactive metabolite acetyl acrolein so far.Thus, it remains to be established if Lys-AcA and AcLys-AcA provide reliable biomarkers to monitor exposure to 2-methylfuran.Controlled exposures in experimental animals in analogy to our present study may be valuable to understand if significant background levels of these metabolites occur.
Overall, our data support the use of GSH-BDA for monitoring furan exposure but highlight significant limitations of NAcLys-BDA, NAcCys-BDA-NAcLys and NAcCys-BDA-NAcLys sulfoxide due to their high background in urine.Future work is needed to confirm GSH-BDA as a specific biomarker of human exposure to furan via food and to assess if analysis of urinary GSH-BDA may provide reliable exposure estimates.

Fig. 4
Fig.4Linear correlation between external [ 13 C 4 ]-furan dose and 24 h urinary excretion of furan metabolites in male (A) and female (B) rats.Data are presented as mean ± standard deviation (n = 5)

Table 1
Semipreparative HPLC methods for purification of synthesized reference substances and internal standards

Table 5
Probable daily furan intakes (PDIs) corresponding to background excretion of furan metabolites [µg/24 h] based on relative excretion rates of the respective [ 13 C 4 ]-furan derived metabolites PDIs were calculated using the following relative 24 h-excretion rates: a Mean relative 24 h excretion rate across dose groups 1-1000 µg/kg bw b Relative 24 h excretion rate of highest dose group 1000 µg/kg bw c Mean relative 24 h excretion rate across dose groups 10-1000 µg/kg bw d PDI calculation: Probable daily intake (μg/kg bw/d) =