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Comparative metabolism of 2-monochloropropane-1,3-diol (2-MCPD) and 3-monochloropropane-1,2-diol (3-MCPD) in rats and humans (Metabolismus von chlorierten Propandiolen in Ratte und Mensch)


Funding programme / funding institution: Deutsche Forschungsgemeinschaft e.V. (DFG) Bonn - Deutschland

Grant number: MO 2520/2-1 - Project number 468535752

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Project description:


Fatty acid esters of 2-monochloropropane-1,3-diol (2-MCPD) and 3-monochloropropane-1,2-diol (3-MCPD) are common heat-induced food contaminants, which are hydrolyzed efficiently in the gastrointestinal tract to release 2/3-MCPD. The International Agency for Research on Cancer (IARC) has classified 3-MCPD as possibly carcinogenic to humans. The TDI of 3-MCPD (2.0 µg kg/body weight) is only about two times higher compared to the median intake levels in infants (0.9 µg kg/body weight per day) and it is exceeded by infants receiving exclusively formula (mean exposure: 2.4 µg/kg body weight per day).

The concepts of the molecular mechanisms explaining the toxicity and carcinogenicity of 2/3-MCPD in rats are limited. A better understanding depends on the available information on the metabolism of both compounds, which may also help to clarify the relevance of some of the toxic effects observed in rats for human health. The current proposal aims at the comparative elucidation of the metabolism of 2/3-MCPD in rats and humans. The animal model allows generating sufficient amounts for a systematic characterization of metabolites. The rats will receive single oral doses of 2- or 3-MCPD and also of [13C3]2-MCPD or [13C3]3-MCPD. The urinary metabolites will be separated by preparative chromatography and characterized by mass spectrometry (MS) and 13C-nuclear magnetic resonance (NMR) spectroscopy. The data on the compounds resulting from rat metabolism will be the basis for the characterization of 2/3-MCPD metabolites excreted in human urine samples, collected in a controlled exposure study (conducted previously) with hazelnut oil containing relatively high amounts bound 2-MCPD (24.2 mg/kg) and 3-MCPD (54.5 mg/kg).

The information of the 2/3-MCPD metabolism is a good basis for the choice of human biomarkers of exposure for both substances. We propose to develop analytical techniques for the quantification of metabolites of 2/3-MCPD in 24-h urine, which meet the requirements on sensitivity and specificity. The selected biomarkers will be used to estimate the external 2/3-MCPD exposure of study participants with distinct dietary habits (omnivores, vegans and raw food eaters; sample collection finished in 2020). In summary, the data provided on 2/3-MCPD metabolism in rats and in humans will help to understand the toxic effects in rats and, possibly, the relevance for humans, and will support determining the human exposure in individuals and future recommendations on risk management.

Specific Aims

  1. Identification of individual metabolites of 2- and 3-MCPD in rats.
  2. Identification of individual metabolites of 2- and 3-MCPD in humans following uptake of contaminated hazel nut oil.
  3. Development of isotope-dilution LC-MS/MS methods for the quantification of the main human metabolites of 2- and 3-MCPD and testing their applicability as biomarkers of exposure.

Aim 1: Identification of individual metabolites of 2- and 3-MCPD in rat urine after oral administration of 2- or 3-MCPD and [13C3]2- or [13C3]3-MCPD

1.1       Strategy

Wistar rats will be treated with single oral doses of 2- or 3-MCPD and the respective 13C-labeled compounds. Urine samples will be collected over 48 h and the metabolites will be identified by MS and NMR spectroscopy. As described below, the 13C-labeling supports identification, structural characterization and quantification of MCPD-specific metabolites by MS and 13C-NMR spectroscopy. The stepwise work plan is as follows.

S1) A survey of urinary metabolites will be performed by LC-MS and LC-MS/MS to generate an inclusion list with possible urinary metabolites of 2- and 3-MCPD.

S2) The urine samples are fractionated by preparative reversed-phase (RP) high-performance liquid chromatography (HPLC) to deplete urinary matrix and to separate the metabolites.

S3) The molecular structures of 2- and 3-MCPD metabolites will be elucidated using MS and 13C-NMR spectroscopy.

S4) The 2- and 3-MCPD metabolites in fractions of rat urine samples will be quantified using 13C-NMR spectroscopy.

1.2       Animal experiment: Administration of 2- and 3-MCPD to Wistar rats

Fifty-six Wistar rats (7 to 8 week old, body weight ∼ 250 g) are acclimatized for five days and then placed into metabolic cages. The animals receive single oral doses of 2- or 3-MCPD dissolved in water or the respective isotope-labeled compounds per gavage. The inclusion of 13C3-labeled compounds allows NMR-based structure elucidation. Urine samples are collected after 24 h and 48 h and stored at - 80°C.

1.3       Mass spectrometric identification of metabolites of 2- and 3-MCPD in urine samples (S1)

Initial analyses of urine samples using LC-MS have two objectives. The main goal is the collection of exact masses and fragmentation patterns of the metabolites of 2- and 3- MCPD. In addition, we will test different conditions to optimize the chromatographic separation of the metabolites. The equipment consists of an UltiMate™ 3000 UHPLC connected to an Orbitrap Q Exactive Focus system (Thermo Fisher).

1.4       Fractionation of 2- and 3-MCPD metabolites with preparative HPLC (S2)

The fractionation (e.g. using a custom-made column with HSS T3 material (19 x 250 mm, Waters)) aims at the depletion of the urinary matrix and the best isolation of the metabolites with highest purity possible, which will facilitate the subsequent characterization by NMR spectroscopy. Fractions obtained are analyzed using an I-Class UPLC system (Waters) and a HSS T3-column (2.1 x 100 mm, Waters) connected to the tandem-quadrupole mass spectrometer QTRAP6500 (Sciex) and concentrated under reduced pressure.

1.5       Structural characterization of metabolites of [13C3]2- and [13C3]3-MCPD by 13C-NMR spectroscopy (S3)

The structural information obtained by MS will be complemented with the analyses of isotope-labeled metabolites by 13C-NMR spectroscopy, a technique that was applied as well to characterize metabolites of acrylonitrile and acrylamide. Natural compounds (1.1% abundance of 13C) give rise to single resonances of relatively low intensity. In contrast, the presence of urinary metabolites of [13C3]2-MCPD and [13C3]3-MCPD (with > 98% 13C) will yield intense signals, which predominate the spectra of single fractions. The 13C-NMR spectra are recorded using a VNMRS 500 MHz spectrometer (Varian) at the Bundesanstalt für Materialforschung und -prüfung (BAM), Fachbereich 1.4 Prozessanalytik (Dr. Maiwald) in Berlin.

1.6       Quantification of [13C3]2- and [13C3]3-MCPD and their metabolites in rat urine (S4)

Quantitative NMR (qNMR) spectroscopy has become an important tool for content determination of organic substances also in metabolomics. Here, we will apply 13C-NMR spectroscopy to determine the concentrations of the metabolites in the HPLC fractions of urine samples from rats treated with [13C3]2-MCPD and [13C3]3-MCPD (section 1.4), which then will be used to calculate the overall amount of the urinary metabolites.

Urinary 2- and 3-MCPD and their metabolites 2-ClHA and 3-ClLA will be quantified directly using isotope-dilution GC-MS and LC-MS/MS.

1.7       Anticipated outcome

The application of relatively high amounts of 2- and 3-MCPD as well as their isotope-labeled congeners in the animal experiment allows identifying the most abundant metabolites in the urine samples. MS and NMR spectroscopy yield a comprehensive data set for the structural characterization. Taken together, two goals shall be reached: the incomplete schemes of 2- and 3-MCPD metabolism in rats will be completed. The amounts of the metabolites excreted will reflect the contribution of different metabolic pathways. Importantly, the data will indicate whether 2- and 3-MCPD metabolism are overlapping. The second goal are two small compound libraries of rat metabolites of 2- and 3-MCPD in solutions with defined concentrations that exist in their natural form and in addition as 13C-labeled molecules. These will be used for the identification of human metabolites (Aim 2).

Aim 2: Metabolites of 2- and 3-MCPD in human urine samples

2.1       Strategy

A controlled exposure study was performed at the BfR with 12 participants ingesting 12 g of commercially available hazel nut oil containing fatty acid esters of 2- and 3-MCPD. The excreted amounts of 2- and 3-MCPD and of 2-ClHA and 3-ClLA were determined. We propose to identify and quantify other human urinary metabolites of 2- and 3-MCPD. The samples will be analyzedng with mass spectrometric techniques of relatively broad detection width (untargeted, low sensitivity) down to more focused detection methods (targeted, high sensitivity). It is assumed, that human and rat metabolism are alike and that the results of Aim 1 can be used as a basis for the identification of 2- and 3-MCPD metabolites in human urine samples. The steps are as follows:

S1) Single human metabolites will be identified and a list containing retention times, exact masses and mass spectrometric fragmentation patterns will be assembled.

S2) The amounts of single metabolites in human urine samples will be determined by isotope-dilution LC-MS/MS.

2.2       Mass spectrometric identification of metabolites of 2- and 3-MCPD in human urine samples (S1)

Sample preparation: The identification in human samples will require enrichment of the low-concentrated metabolites and depletion of urinary matrix. We will compare different techniques, because their efficiencies may vary depending on the properties of the target metabolites.

Three approaches of mass spectrometric identification: a) Full-scan spectra. The identification follows a similar strategy as described in section 1.3 using the UltiMate 3000 UHPLC connected to an Orbitrap Q Exactive Focus. b) Targeted MS. The targeted examination of 2- and 3-MCPD metabolites in human urine samples based on the annotation lists of rat metabolites is a more sensitive approach. c) Screening for expected fragmentation patterns (“fixed-step selected ion monitoring”).

2.3       Mass spectrometric quantification of metabolites of 2- and 3-MCPD in human urine samples (S2)

A technique will be developed for the simultaneous detection of 2- and 3-MCPD metabolites identified previously (section 2.2) by LC-MS/MS MRM based on the data on retention times and fragmentation patterns. We expect that rat and human metabolism strongly overlap. This will permit using the libraries of solutions containing the metabolites of [13C3]2-MCPD and [13C3]3-MCPD at known concentrations (Aim 1) as reference standards for the quantification of the human metabolites. The MRM method allows the quantification of the metabolites of 2- and 3-MCPD in all urine samples from the human study (264 samples). The evaluation yields excretion kinetics of all metabolites.

2.4       Anticipated outcome

The quantification of 2- and 3-MCPD and of the metabolites 3-ClLA and 2-ClHA in all urine samples after controlled exposure to hazelnut oil has been completed. The mass spectrometric identification of human metabolites based on the data of rat metabolism (Aim 1) is a promising strategy, which will add to the evolving schemes of human 2- and 3-MCPD metabolism.

Aim 3: Urinary biomarkers of 2- and 3-MCPD exposure

3.1       Strategy

Urinary metabolites are preferred as short-term exposure biomarkers. The data on human metabolites (Aim 2) will allow selecting candidate compounds for the exposure estimation, which meet the criteria of sensitive detection and specificity for 2-MCPD or 3-MCPD. The work flow is summarized as follows:

S1) The specificity of the biomarker candidates, a prerequisite for their use to determine the exposure of 2- and 3-MCPD by reverse dosimetry, will be assessed.

S2) The analytical process devised before (section 2.3) will be optimized for the quantification of the targeted biomarkers by isotope-dilution LC-MS/MS.

S3) The biomarkers will be used for the calculation of 2- and 3-MCPD exposure using 24-h urine samples from two studies on health effects of dietary habits currently ongoing at the BfR.

3.2       Evaluation of the specificity of the biomarker candidates (S1)

The selection of candidate biomarkers for 2- and 3-MCPD exposure is based on the previous results on human metabolism (section 2.3). As the specificity of the biomarker candidates is crucial for the application in reverse dosimetry, we will study several aspects to clarify this point.

3.3       Optimization of biomarker quantification

Another primary criterion of biomarker selection is the abundance of the urinary metabolites and the expected feasibility of analytical detection. For the quantification, stable isotope-labeled standard substances of the target analytes will be purchased from companies offering custom synthesis. We will re-evaluate the analytical methods devised in Aim 2 (section 2.3) and, if needed, optimize the urinary extraction of the target compounds (recovery), the chromatographic separation and the mass spectrometric quantification by MRM.

3.4       Assessment of external exposure to 2- and 3-MCPD in groups of adult omnivores, vegans and rawfood eaters (S3)

After the selection of specific and sufficiently abundant metabolites of 2- and 3-MCPD as biomarkers, the optimized analytical techniques will be applied to the exposure assessment in participants with different dietary habits (omnivores, vegans and raw food eaters) from two studies on nutrition and health currently in progress at the BfR.

3.5       Anticipated outcome

Various compounds considered previously as urinary biomarkers of exposure to the fatty acid esters of 2- and 3-MCPD were lacking sensitivity and/or specificity. The proposed work allows detecting novel urinary biomarkers for 2- and 3-MCPD exposure, and the development of suitable mass spectrometric methods for their quantification in human urine. If the exploration on biomarkers is not successful, we may use 2-MCPD and 3-MCPD themselves as urinary biomarkers in order to study the external exposure in the current studies with omnivores, vegans and raw food eaters at the BfR (S3). It was discussed that the sensitivities of detection are a limiting factor for future human biomonitoring with urinary 2- and 3-MCPD. The excretion of the biomarkers determined in 24-h urine samples of the study participants (S3) allows estimating the daily exposure to 2- and 3-MCPD among omnivores, vegans and raw food eaters. Detailed food intake data is available. Correlations between food classes and biomarker excretion may elucidate main sources of the 2- and 3-MCPD exposure in the study participants. It is of particular interest, whether the biomarkers of 2- and 3-MCPD exposure will be detectable in raw food eaters at all because they do not consume refined oils.

Project partners

  • Fachbereich 1.1 - Anorganische Spurenanalytik (BAM) (Bundesanstalt für Materialforschung und -prüfung)
  • SGS Germany GmbH (SGS) - Deutschland


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