Toxicokinetics and metabolism of 3-monochloropropane 1,2-diol (3

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Bioactive Constituents, Metabolites, and Functions

Toxicokinetics and metabolism of 3-monochloropropane 1,2-diol (3-MCPD) dipalmitate in Sprague Dawley rats Guoren Huang, Boyan Gao, Jinli Xue, Zhihong Cheng, Xiangjun Sun, Yaqiong Zhang, and Liangli (Lucy) Yu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05422 • Publication Date (Web): 10 Oct 2018 Downloaded from http://pubs.acs.org on October 14, 2018

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Journal of Agricultural and Food Chemistry

Toxicokinetics and metabolism of 3-monochloropropane 1,2-diol (3-MCPD) dipalmitate in Sprague Dawley rats Guoren Huang1, 2, Boyan Gao1, 2*, Jinli Xue2, Zhihong Cheng3, Xiangjun Sun2, Yaqiong Zhang2, Liangli (Lucy) Yu4*

1

Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing

Technology & Business University (BTBU), Beijing 100048, China 2

Institute of Food and Nutraceutical Science, School of Agriculture & Biology, Shanghai Jiao

Tong University, Shanghai 200240, China 3 Department

of Pharmacognosy, School of Pharmacy, Fudan University, Shanghai 201203,

China. 4

Department of Nutrition and Food Science, University of Maryland, 0112 Skinner Building,

College Park, MD 20742, USA

* Contact Information of the Corresponding Author: Liangli (Lucy) Yu, Tel: 301-0405-0761, Fax: 301-314-3313, Email: [email protected]; Correspondence may also be directed to Boyan Gao, Tel: 86-3420-4538, Email: [email protected]

ACS Paragon Plus Environment


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Abstract

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Fatty acid esters of 3-monochloropropane 1,2-diol (3-MCPD) are a group of

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processing-induced toxicants. To better clarify their possible toxicological effects and

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mechanisms, it is important to investigate their absorption, distribution, metabolism and

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excretion. In this study, the kinetic parameters of 3-MCPD dipalmitate in Sprague Dawley (SD)

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rat plasma were determined using ultraperformance liquid chromatography-triple

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quadrupole-mass spectroscopy (UPLC-TQS-MS). 3-MCPD dipalmitate was absorbed in rats

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with a Cmax of 135.00 ng/mL, T1/2 of 3.87 h, Tmax of 2.5 h, MRT of 5.08 h, CL of 3.50 L/h/g, Vd

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of 21.34 L/g, and AUC0-∞ of 458.47 hng/mL. A total of 17 metabolites were identified, and 16 of

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that were reported for the first time. Furthermore, these metabolites were examined for their

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presences in the liver, kidney, testis, brain, spleen, thymus, intestine, plasma, faeces and urine

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samples 2, 6, 12, 24 and 48 hours after oral administration of 3-MCPD dipalmitate using a

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Metabolynx software.

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Key words: 3-MCPD dipalmitate; toxicokinetics; UPLC-MS; absorption, distribution,

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metabolism and excretion (ADME).


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Introduction The fatty acid esters of 3-monochloropropane 1,2-diol (3-MCPD esters) are a group of food

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contaminants formed during food processing in a range of 0.14-27 mg/kg in many food

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categories and food ingredients, especially in refined edible oils.1-5 3-MCPD esters have become

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a real food safety concern due to their toxicities.6-9 Kidney is the primary target organ of

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3-MCPD esters.6-10 3-MCPD esters caused kidney tubular necrosis and epididymis epithelial

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apoptosis at doses of 22.5-360 μmol/kg BW/day in F344 rats in a 13-week toxicological study.7

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3-MCPD esters also induced kidney tubular cell apoptosis in a single dose of 1000 mg/kg BW in

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Sprague Dawley (SD) rats.11 Moreover, 3-MCPD dipalmitate at a concentration of 9.78-156.75

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mg/kg BW/day was able to induce tubular and testicular toxicities in Wistar rats in a 90-day

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toxicological study.10 In addition, 3-MCPD dipalmitate, 3-MCPD distearate, 3-MCPD dioleate,

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and 3-MCPD dilinoleate induced lipid metabolism disorders in C57BL/6J mice at doses of 3, 6

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and 11 mg/kg BW/day.12 Recently, Liu and colleagues reported that 3-MCPD esters could

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induce acute toxicity in brain, thymus and lung in Swiss mice at a treatment level of 4162 mg/kg

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BW.9

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The toxicity of 3-MCPD esters was partly attributed to 3-MCPD, which might be released

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from 3-MCPD esters in Caco-2 cells, human and Wistar rats.13-17 In addition, Seefelder and

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colleagues reported that 3-MCPD monoesters were effectively hydrolyzed to free 3-MCPD by

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lipase from porcine pancreas in vitro.18 Moreover, the metabolic rate of 3-MCPD diesters to

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3-MCPD was much slower compared with that of monoesters in vitro.13, 18 Abraham and others 3



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reported that the diester form of 3-MCPD had 86% bioavailability compared with free 3-MCPD

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in Wistar rats at a single dose of 53.2 mg/kg BW,14 while Barocelli and others demonstrated that

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the bioavailability of 3-MCPD dipalmitate was 70% of the free 3-MCPD in Wistar rats a dose of

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156.7 mg/kg BW under the experimental conditions.10 Barocelli et al. also reported that free

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3-MCPD and its metabolites 2,3-dihydroxypropyl mercapturic acid (DHPMA) and β-chlorolactic

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acid were the metabolites of 3-MCPD dipalmitate.10 However, these previous studies primarily

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focused on free 3-MCPD, but not the other possible metabolites of 3-MCPD esters in vivo. In

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addition, the bioavailability results could not explain the difference in the toxic effects between

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3-MCPD monoester and diester in Swiss mice or SD rats on an equimolar basis.6, 9, 11 Recently,

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Gao and others reported that 8 metabolites of 3-MCPD 1-palmitate after a single oral dose of 400

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mg/kg BW were found in rats, including 3-MCPD, sulfonated 3-MCPD, acetylated 3-MCPD,

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glucuronidated 3-MCPD, and 3-MCPD bonded with different amino acids, and the major

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excretion of 3-MCPD 1-palmitate was suggested through urine.19 It is interesting whether and

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how 3-MCPD diesters can be absorbed, distributed to different tissues and organs, metabolized

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and excreted.

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The present study was conducted to investigate the toxicokinetics of 3-MCPD dipalmitate at

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a dose of 1600 mg/kg, and to characterize 3-MCPD dipalmitate metabolites and their distribution

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in liver, kidney, testis, spleen, intestine, thymus, brain tissues as well as in faecal and urine

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samples with two animal feeding studies in SD rats. A possible metabolism pathway(s) in SD rat

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model was also suggested. The treatment dose was selected as a possible greatest dose based on 4


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the preliminary experiments and previous studies, so that more metabolites could reach their

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limit of detection (LOD) of the analytical method and could be detectable under the experimental

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conditions. This is the first study on the absorption, distribution, metabolism and excretion

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(ADME) of 3-MCPD dipalmitate. The results may contribute to overall knowledge of the organ

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specific toxicities of 3-MCPD diesters.

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Materials and Methods

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Chemicals and reagents. 1,2-Bis-palmitoyl-3-chloropropanediol (3-MCPD dipalmitate),

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1-Palmitoyl-3-chloropropanediol (3-MCPD 1-palmitate) and 2-Palmitoyl-3-chloropropanediol

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(3-MCPD 2-palmitate) were synthesized in the lab. And their purity was above 98% tested by

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ultraperformance liquid chromatography-quadrupole time of flight-mass spectroscopy

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(UPLC-QTOF-MS) and nuclear magnetic resonance (NMR). The standard 3-MCPD dipalmitate

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and the deuterated internal standard rac 1,2-Bis-palmitoyl-3-chloropropanediol-d5 (3-MCPD

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d5-dipalmitate) were purchased from Toronto Research Chemicals (Ontario, Canada). Liquid

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chromatography mass spectroscopy (LC-MS) grade isopropanol and sodium acetate were

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purchased from Sigma-Aldrich (St. Louis, USA). LC-MS grade methanol was purchased from

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CNW Technologies (Düsseldorf, Germany). LC-MS grade water was obtained from a Milli-Q 10

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ultra-pure water system (Billerica, USA). All the other chemical reagents were analytical grade,

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purchased from Sigma-Aldrich (St. Louis, USA) and used without further purification.

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Animals, diet and housing conditions. Male SD rats (160 ± 20 g, 6-8 weeks old) were purchased from Shanghai SLAC Laboratory Animal Co. Ltd (Shanghai, China). The rats were 5



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maintained in a temperature controlled room maintained on a 12 h light/dark cycle, and were

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allowed free access to drinking water and standard diet (including approximately 5% clued oil,

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XIETONG, Jiangsu, China).19, 20 The animals were acclimatized to the facilities for a week, and

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then fasted with free access to water for 12 h prior to experiment. All procedures involving

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animal uses were conducted in accordance with the Animal Care and Welfare Committee of the

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Laboratory Animal Center of Shanghai Jiao Tong University with a certification number of

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A2015024.

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Animal treatment and sample collection. 3-MCPD dipalmitate was orally administrated

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to rats. After 4 hours, the water and food were fed ad libitum. Seventy-two SD rats were

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separated into 12 groups randomly, and each group had six rats. Two groups were used for

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toxicokinetics analysis. After administration of 3-MCPD dipalmitate (test group) or extra virgin

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olive oil free of 3 MCPD esters (control group), blood samples were collected in heparinized

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Eppendorf tube via the oculi chorioidea vein before dosing and subsequently at sixteen time

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points (0, 0.167, 0.33, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 12, 24, and 48 h).19, 21

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The rest ten groups of rats were used for the metabolic studies of 3-MCPD dipalmitate.

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Briefly, five groups were kept in metabolic cages, and the SD rats were anaesthetized with CO2

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and decapitated to collect the liver, kidney, testis, spleen, intestine, thymus and brain at 2, 6, 12,

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24 and 48 hours after oral administration of 3-MCPD dipalmitate. The faeces and urine samples

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were collected in metabolic cages at different time points. For instance, urine samples at 12

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hours represented the urine collected between 10 and 12 hours. Another five groups were set as 6


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control groups, oral administration of equal amount of extra virgin olive oil free of 3-MCPD

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esters, and sacrificed at similar five time points as the five test groups. All biological samples

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except blood samples were rapidly frozen at -80 °C until analysis.

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Preparation of animal samples. The animal samples were prepared according to

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previously reported protocols.19 Briefly, blood samples were centrifuged, and the supernatant

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plasma was collected and rapidly frozen at -80 °C until assay. 300 µL of acetonitrile was added

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to each 100 µL of plasma sample, and followed by vortexing and centrifugation at 9391 g for 10

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minutes at 4 °C. The supernatant was transferred into a UPLC vial. 0.1 g of each liver, kidney,

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testis, spleen, intestine, thymus, or brain tissue sample was homogenized with 900 μL ice-cold

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saline at 4 °C. After 900 μL acetonitrile was added to 300 µL of the tissue homogenate, each

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mixture was vortexed and centrifuged at 9391 g at 4 °C for 10 min, and the supernatant was

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collected and transferred to an UPLC vial. In addition, water-saturated ethyl acetate was used to

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extract urine sample at a solvent to urine ratio of 5/1 (v/v), and the solvent was removed from the

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supernatant by evaporating using a nitrogen evaporator at ambient temperature. The residue was

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re-dissolved with methanol. 0.1 g of the freeze-dried faeces sample was extracted by 1.5 mL

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ethyl acetate for two times, and the solvent was removed from the supernatant using a nitrogen

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evaporator after centrifugation. The residue was re-dissolved with methanol.

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UPLC-MS analyses. A Waters Acuity UPLC-TQS-MS (Waters, Milford, MA, USA) with

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a Waters Phenyl column (2.1 mm i.d. × 50 mm, 1.7 μm) was selected to quantify 3-MCPD

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dipalmitate levels in rat plasma. The mobile phase consisted of (A) water/methanol (9:1, v/v) 7



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with 1 mg/L NaOAc, and (B) methanol/ isopropanol (4:1, v/v) with 1 mg/L NaOAc, using the

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elution gradient beginning with 35% phase B, changed linearly to 95% B in 3 min and

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maintained for 2 min, and returned to its initial conditions for 1 min to re-equilibrate the column

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for the next injection. The flow rate was 0.4 mL/min with an injection volume of 2 μL. The MS

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detector conditions were: capillary voltage 3.50 kV; sampling cone voltage 35 V; extraction cone

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voltage 4.0 V; source temperature 120 °C; and desolvation temperature was 450 °C. The cone

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gas flow rate was 150 L/h and the desolvation gas was 1000 L/h. An ion pair with a precursor ion

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at m/z 609.4627 ([M+Na]+) and a product ion at m/z 551.5026 for 3-MCPD dipalmitate, and m/z

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371.2329 ([M+Na]+) and a product ion at m/z 313.2747 for 3-MCPD 1-palmitate and 3-MCPD

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2-palmitate were selected to create a multiple reaction monitoring (MRM) method with the

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collision energy of 35 eV in ESI positive mode.

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A Waters Acuity UPLC-Xevo G2-QTOF-MS system (Waters, Milford, MA, USA) was

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selected for metabolite identification with a Waters Phenyl column (2.1 mm i.d. × 100 mm, 1.7

130

μm). The mobile phase consisted of (A) water/methanol (9:1, v/v)) with 1 mg/L NaOAc, and (B)

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methanol/ isopropanol (4:1, v/v) with 1 mg/L NaOAc, and using the elution gradient started with

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0% phase B, changed linearly to 35% B in 4 min, then changed linearly to 95% B in another 4

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min and maintained for 2 min, and returned to its initial conditions and kept for 2 min to

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re-equilibrate the column for the next injection. For MS detector conditions, capillary voltage

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3.00 kV; sampling cone voltage 35 V; extraction cone voltage 4.0 V; source temperature 120 °C;

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and desolvation temperature 450 °C. The cone gas flow rate was 150 L/h and the desolvation gas 8


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was 800 L/h. A MSE method was used with mass range from 50 to 1200 m/z in both ESI positive

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and ESI negative mode, scan time was 0.3 s and the ramp collision energy was 20-35 eV.

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The Waters Acquity Ultra Performance Convergence Chromatography™ (UPCC) equipped

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with TQS-MS was used to separate 3-MCPD 1-palmitate and 3-MCPD 2-palmitate. The

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qualitative analysis was performed at 50 °C using an Acquity UPCC HSS C18 SB column (100

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mm × 3.0 mm i.d.; 1.8μm; Waters, Milford, MA, USA). The elution gradient (solvent A, CO2;

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solvent B, isopropanol: acetonitrile at 1:1, v/v) was started at 0.2% B; increased via linear

144

gradient to 20% B at 4 min, and maintained for 1 min, and returned to its initial conditions for 1

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min. The back pressure was set at 1600 psi. The flow rate was 1.4 mL/min and the injection

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volume was 2.0 μL. MS detector conditions were same as UPLC-TQS-MS.

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Method development and validation. Current UPLC conditions and MS parameters were

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validated as described in a previous study.19 Briefly, different mobile phases and MS parameters

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were tested in the UPLC-MS system to select the appropriate mobile phase and MS method.

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3-MCPD d5-dipalmitate was used as the internal standard. And the LOD and limit of

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quantification (LOQ), relative standard deviation (RSD), standard curve and linear range, and

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recovery were validated as described by Gao and others .19 The LOD and LOQ in this study were

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0.3 ng/mL and 1 ng/mL of 3-MCPD dipalmitate, respectively. The RSD was 1.82-4.07%. The

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calibration curve of the 3-MCPD dipalmitate was linear in the range of 10-1000 ng/mL and the

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correlation coefficient was 0.9994. The recovery of 3-MCPD dipalmitate in plasma samples was

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93.00 to 109.66%. 9



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GC-MS analyses. Free 3-MCPD was identified by a modified DGF method with GC-MS.

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Brief, approx. 0.1 g tissue samples with 0.5 mL t-butyl methyl ether (tBME)/ethyl acetate (4:1,

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v/v) were homogenize at 4 °C, and kept in an ultrasonic bath at 45 °C (starting temperature) for

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15 min to remove glycidol. After adding 3 mL of hexane and 3 mL of saturated sodium sulfate,

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the organic phase was removed with a pipette and discarded. After the addition of another 3 mL

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hexane the mixture was shaken carefully. After the separation of the phases the upper hexane

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phase was completely withdrawn with a pipette and discarded. 500 μL of the derivatization

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reagent (Phenylboronic acid) was added into the aqueous phase, and reacted at 80 °C for 20

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minutes. After cooling to ambient temperature, the 3-MCPD derivatives were extracted with 3

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mL hexane for GC-MS analysis. GC-MS (7890A-5975C, Agilent Technologies, Santa Clara,

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USA) instrument with a DB-5MS（30 m × 0.25 mm, 0.25 µm, Agilent Technologies, Santa

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Clara, USA) was used with the following settings: column temperature, 270 °C (1 min hold),

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20 °C/min to 300 °C and hold for 5 min; carrier gas, He; constant linear velocity, 37 cm/s; inlet

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temperature, 300 °C; injection mode, splitless; and injection volume, 1 μL. MS was operated in

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the EI mode (70 eV, 60 μA) with the following settings: ion source temperature, 280 °C; mode,

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SIM; monitoring ions, m/z 196, 147, 91 for 3-MCPD.26

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Data and statistical analyses. Results were expressed as mean ± standard deviation. The

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kinetics of 3-MCPD dipalmitate in SD rat plasma was analyzed using Phoenix WinNonlin

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software for Windows (version 6.4, Pharsight, Princeton, NJ). Toxicokinetics parameters

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including peak plasma concentration (Cmax), time to reach Cmax (Tmax), elimination half-life 10


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(T1/2), mean residence time (MRT) were obtained directly from the plasma -time data. The

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clearance rate (CL), volume of distribution (Vd) and total area under the plasma

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concentration-time curve (AUC0-∞) were calculated using Phoenix WinNonlin software based on

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the trapezoidal rule.

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Results and discussion

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Kinetics of 3-MCPD dipalmitate in rats. The toxicokinetics parameters including Cmax,

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Tmax, T1/2, MRT, CL, Vd and AUC0-∞ are reported in Table 1. 3-MCPD dipalmitate was detected

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in the plasma of SD rats at 20 min (data not shown) and reached Cmax of 135.00 ng/mL at about

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2.5 h (Tmax) after a single oral gavage of 1600 mg/kg 3-MCPD dipalmitate. The Tmax was longer

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than that for free 3-MCPD of 30 min14 and the Tmax of 1.67 h for 3-MCPD 1-palmitate.19 In the

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present study, the T1/2, MRT, CL and Vd were 3.87 h, 5.08 h, 3.50 L/h/g and 21.34 L/g,

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respectively. The average AUC0-∞ for 3-MCPD dipalmitate in SD rat plasma was 458.47

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hng/mL. These data suggested that 3-MCPD dipalmitate could be absorbed in body circulation

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as its diester form, which was supported by the observation in a previous study that 3-MCPD

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dipalmitate was detected in the blood of F344 rats 20 and a previous report that 3-MCPD

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dipalmitate was detected in human breast milk.22 In the study using F344 rats, a one-time-point

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absorption of 3-MCPD dipalmitate at 30 min was reported at 0.06 nmol/mL (~35 ng/mL) after

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treated with 360 μmol/kg BW 3-MCPD dipalmitate, which was about 1/4 of the Cmax value

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compared with that from the present study.20 In contrast, two previous studies did not detect

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original form of 3-MCPD dipalmitate in the plasma samples of Wistar rats at doses of 156.75 11



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mg/kg BW and 53.2 mg/kg BW of 3-MCPD dipalmitate in 24 h after oral intake.10, 14 The

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differences in 3-MCPD dipalmitate absorption may be partially explained by the animal species,

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body weight and age of the animals, dose of 3-MCPD dipalmitate, and sample analysis

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techniques. Additional studies are needed to further investigate the absorption of 3-MCPD

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diesters in more experimental animal models and using diesters with different fatty acids.

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It needs to be pointed out that the treatment dose of the present study is much greater than

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the level of 3-MCPD esters that people may possibly obtain from food samples. It also needs to

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realize that there is no information available on whether 3-MCPD esters might be accumulative

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in human and animal bodies. The purpose of the present study was to investigate possible

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absorption, distribution, metabolism and excretion of 3-MCPD esters. A relatively greater

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concentration used in the study could provide more information about the potential metabolites

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of 3-MCPD dipalmitate as well as the metabolic parameters in this animal model.

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Fragmentation patterns of 3-MCPD esters. The fragmentation patterns of synthetic

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standard 3-MCPD dipalmitate, 3-MCPD 1-palmitate and 3-MCPD 2-palmitate were examined

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and used as references for identifying the 3-MCPD esters and their metabolites in the plasma and

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tissue samples. Under positive ESI mode, 3-MCPD dipalmitate formed a [M+Na]+ ion at m/z

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609.4627. The high collision energy mass spectrum showed characteristic product ions at m/z

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551 and 573 due to the neutral loss of an HCl unit with or without a Na+ ion (Supplementary

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Figure S1A). Both 3-MCPD 1-palmitate and 3-MCPD 2-palmitate have protonated molecule ion

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[M+Na]+ at m/z 371.2329, and could yield a product ion at m/z 313 due to the neutral loss of an 12


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HCl unit with a Na+ ion (Supplementary Figure S1B, C). To separate 3-MCPD 1-palmitate

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from 3-MCPD 2-palmitate, a UPCC-TQS-MS method was developed and utilized. UPCC is a

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supercritical fluid chromatography (SFC) which is suitable for non- and low-polarity compound

220

separation.23, 24 As shown in Supplementary Figure S2, 3-MCPD 1-palmitate and 3-MCPD

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2-palmitate were successfully separated from each other with retention time of 2.62 min and 3.06

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min, respectively. Taken together, the information about these fragmentation patterns was

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helpful in characterizing and identifying the prototype and major metabolites of 3-MCPD

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dipalmitate in vivo.

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Identification of 3-MCPD dipalmitate metabolites. A total of 17 metabolites of 3-MCPD

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dipalmitate were identified or tentatively identified in the SD rat plasma, urine, faeces, liver,

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kidney, testis, spleen, intestine, thymus and brain samples collected at 2, 6, 12, 24 and 48 h after

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3-MCPD dipalmitate administration based on their accurate formula masses and fragment ion

229

patterns using Waters MetaboLynx software. Metabolites included 3-MCPD 2-palmitate (M1),

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glycidol palmitate (M2), 3-MCPD palmitoleate-palmitate (M3), 3-MCPD palmitate-sterate (M4),

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3-MCPD palmitate-oleate (M5), 3-MCPD palmitate-linoleate (M6), 3-MCPD

232

palmitate-arachidonate (M7), 3-MCPD sterate (M8), 3-MCPD oleate (M9), 3-MCPD linoleate

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(M10), 3-MCPD arachidonate (M11), glucuronidated 3-MCPD 2-palmitate (M12), glutathione

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conjugate of monopalmitate (M13), palmitic acid (M14), sulfonated dipalmitate (M15),

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glucuronided 3-MCPD (M16) and 3-MCPD (M17), along with 3-MCPD dipalmitate (Figure 1).

236

M17 was identified by GC-MS method, whereas the other metabolites and 3-MCPD dipalmitate 13



237

were identified based on UPLC-QTOF-MS analysis. It needs to point out that this study did not

238

quantify these metabolites in plasma or tissue samples. The chemical formulas, accurate masses,

239

retention time and mass fragments are summarized in Table 2 and Supplementary Figure

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S3-S7.

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Metabolite M1 (Figure 2) was selected as an example to demonstrate how these metabolites

242

were identified. M1 had a protonated molecule [M+Na]+ at m/z 371.2329 (Table 2, Figure 3A).

243

The major fragment of M1 under a stronger collision energy was obtained at m/z 313.2730

244

(Table 2, Figure 3B), 238 Da less than that of 3-MCPD dipalmitate (M0) (Supplementary

245

Figure S1A), indicating a possible loss of palmitic acid. UPCC-MS was employed to further

246

reveal the cleavage position of the palmitic acid. By comparing the UPCC-MS characteristic

247

spectra of M1 with the synthetic standard 3-MCPD 1-palmitate and 3-MCPD 2-palmitate, the

248

metabolite M1 in the liver and intestine with a retention time of 3.06 min was identified as

249

3-MCPD 2-palmitate (Supplementary Figure S2). No 3-MCPD 1-palmitate was detected in

250

rats’ tissues or plasma. These data suggested that sn-1 palmitic acid of 3-MCPD dipalmitate was

251

hydrolyzed possibly with a pancreatic lipase to generate a sn-2 3-MCPD monoester (M1) that

252

was absorbed into circulation system.17, 18 Due to the lack of standards, the fatty acid sn-

253

locations in the M3-M11 3-MCPD esters could not be identified, and the possible structures of

254

M3-M11 are provided in the dashed frames in Figure 1.

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Glycidyl ester was identified as a metabolite of 3-MCPD dipalmitate in SD rat (Table 2). This observation was supported by a previous report that glycidol was detected in the serum of 14


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the F344 rats given 3-MCPD dipalmitate or 3-MCPD dioleate20. Interestingly, diesters of

258

3-MCPD were detected with one of the two palmitic acids substituted by other fatty acids (Table

259

2). To the best of our knowledge, this is the first report about the transesterification of 3-MCPD

260

diesters in an animal model. Together, these results supported the previous suggestions that

261

3-MCPD diesters could be hydrolyzed at the sn-1 position firstly to a 3-MCPD 2-monoester, and

262

form a new 3-MCPD diesters catalyzed by enzyme(s) such as an esterase or lipase, for instance

263

monoacylglycerol acyltransferase or glycerol-3-phosphate acyltransferase.13, 17, 18, 25 Gao and

264

colleagues reported that 3-MCPD 1-palmitate was metabolized through acetylation,

265

glucuronidation and sulfonation in SD rats.19 In the present study, the identified metabolites such

266

as glucuronidated 3-MCPD 2-palmitate (M12), glutathione conjugate of monopalmitate (M13),

267

sulfonated dipalmitate (M15) and glucuronided 3-MCPD (M16) are the typical phase II

268

metabolites in vivo. By conjugating with the functional groups, the water solubility of these

269

metabolites could be increased, which might accelerate their distribution and excretion.19

270

Glucuronidated 3-MCPD (M16) and free 3-MCPD (M17) were the common metabolites of

271

3-MCPD dipalmitate and 3-MCPD 1-palmitate,19 suggesting that both 3-MCPD diester and

272

3-MCPD monoerster treatments would lead to 3-MCPD-induced toxic effect in vivo.6,7,9-11

273

Interestingly, DHPMA and β-chlorolactic acid, which were reported in urine as 3-MCPD

274

dipalmitate metabolites by Barocelli and others in Wistar rats with a LC-MS/MS method,10 were

275

not detected in the present study. This might be partially due to the different animal models,

276

samples preparation, analytical instrument resolutions, and data analysis software for the two 15



277

studies,10 warranting additional research to investigate the absorption, distribution and

278

metabolism of 3-MCPD esters.

279

Distribution of 3-MCPD dipalmitate metabolites. The distribution of 3-MCPD

280

dipalmitate and its metabolites in plasma, liver, kidney, brain, testis, thymus, intestine, spleen,

281

urine and faeces were summarized in Figure 4. 3-MCPD dipalmitate was detected in liver,

282

kidney, brain, testis, thymus, intestine, spleen and plasma of SD rats after a single oral dose of

283

1600 mg/kg BW, suggesting that 3-MCPD dipalmitate could be absorbed into the plasma and

284

distributed into tissues in its original form. The distribution of 3-MCPD dipalmitate in tissues,

285

such as kidney, testis, brain and thymus, might explain its specific organ toxicities found in the

286

previous studies.6-11 The UPLC-QTOF-MS chromatogram of 3-MCPD dipalmitate in liver is

287

provided in Supplementary Figure S8. The quantification of 3-MCPD dipalmitate and its

288

metabolites in each organ or tissue samples was not performed due to the lack of standards, as

289

well as the facts that no international standard was included during sample preparation for

290

quantitative analysis, and that the free 3-MCPD was estimated using a GC-MS method. 3-MCPD

291

dipalmitate could be detected in faeces at time point of 2, 6, 12, 24 and 48 h after oral

292

administration, but could not be found in urine (Figure 4). This was supported by Abraham and

293

colleagues’ observation that about 33% of 3-MCPD dipalmitate (53.2 mg/kg BW) was found in

294

faeces as ester form, and their conclusion that 3-MCPD dipalmitate could be excreted via

295

faeces.14

296

Furthermore, M0, M1, M5, M6, M7 and M14 were detected in the plasma for the first time. 16


Page 16 of 37

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297

M0, M1, M2, M5, M6, M7, M12, M13, M14, M15 and M17 were also detected in liver,

298

suggesting that the hydrolysis, transesterification and endogenous substance conjugation reaction

299

might occur in liver. It should be noted that orally administered 3-MCPD dipalmitate and

300

3-MCPD dioleate were metabolized to glycidol in F344 rats,20 suggesting that the similar

301

dechlorination reaction occurred in F344 and SD rats. M0, M1, M2, M5, M6, M12, M13, M14

302

and M17 were detected in kidney, indicating that these metabolites might also contribute to the

303

specific organ toxicity to kidney. M0, M1, M13, M14 and M15 were detected in brain,

304

suggesting their potential to pass through the blood-brain barrier. M0, M1, M5, M13 and M14

305

were detected in testis, suggesting their potential to pass through the blood-testis barrier.

306

3-MCPD dipalmitate and its metabolites were detected in the brain and testis under the

307

experimental conditions which was in a good agreement with the previous reports that 3-MCPD

308

1-palmitate could transport through the blood-brain barrier and blood-testis barrier,20, 27 inducing

309

possible toxicity in central nervous system (CNS) and spermatogenic cells.28 M0, M1, M14 and

310

M15 were detected in thymus, agreeing to the previous observation that apoptotic bodies were

311

found in 3-MCPD diesters treated Swiss mice.9 All of the metabolites except M16 and M17 were

312

detected in intestine. M0, M1, M2, M5, M6, M12, M13 and M14 were detected in spleen,

313

warranting additional research to investigate their possible contribution to immune suppression.

314

These results also supported that the hydrolysis, transesterification, glucuronide conjugation,

315

glutathione conjugation and sulfate conjugation occurred in SD rats. M14, M15, M16 and M17

316

were detected in urine, suggesting that 3-MCPD dipalmitate could be excreted through urine 17



317

after converted to more polar substances. 3-MCPD (M17) was detected in liver, kidney and urine

318

using a modified DGF method of GC-MS.26 The same metabolite (M17) of 3-MCPD dipalmitate

319

had been found in urine of Wistar rats in previous studies.10, 14 M0, M1, M2, M4, M5, M6, M7,

320

M9, M10, M11, M14 and M15 were detected in faeces, indicated another possible excretion way

321

of 3-MCPD dipalmitate and its metabolites.

322

One sn-2 3-MCPD monoester (M1), together with other four possible sn-2 3-MCPD

323

monoesters (M8-M11), were detected in the present study. These metabolites were possibly from

324

the hydrolysis of the 3-MCPD diesters (M0, M4-M7). To date, little is known about the potential

325

toxicological properties of the sn-2 3-MCPD monoesters in vivo, warranting additional studies.

326

In addition, 3-MCPD, its fatty acid esters and glycidyl ester (M0-M11, M16) were detected

327

in tissue and body fluid (Figure 4) at all the five time points except 48 h, suggesting that

328

3-MCPD dipalmitate might be metabolized and excreted between 24 h and 48 h after oral

329

administration of 1600 mg/kg BW 3-MCPD dipalmitate. M12-M15 were detected at all the five

330

time points in tissue and urine (Figure 4), suggesting that the metabolism and excretion of

331

3-MCPD dipalmitate was much slower than 3-MCPD 1-palmitate in SD rats.19 M16 was detected

332

in the urine sample at all the five time points except 48 h.

333

Possible metabolic pathway of 3-MCPD dipalmitate in SD rat. A possible metabolic

334

pathway of 3-MCPD dipalmitate in SD rat was proposed using MetaboLynx software (Figure 4).

335

Briefly, 3-MCPD dipalmitate could undergo an elimination reaction, losing a fatty acid and Cl,

336

to form a glycidyl ester, or be hydrolyzed to form 3-MCPD monoesters and further to free 18


Page 18 of 37

Page 19 of 37


337

3-MCPD (phase I metabolites). The phase I metabolites could further undergo possible

338

conjugation reactions to form more polar metabolites for urine excretion (phase II metabolites).

339

3-MCPD was categorized as a ‘possible human carcinogen’-category 2B by International

340

Agency for Research on Cancer.32 Seefelder and colleagues demonstrated that 95% of 3-MCPD

341

dipalmitate could be hydrolyzed to 3-MCPD in vitro within 90 min, 18 whereas Kaze and

342

colleagues found that the hydrolysis rate of 3-MCPD 2-oleate was 80% slower than 3-MCPD

343

1-oleate by the pancreatic lipase in vitro.17 Moreover, an in vivo experiment using a rat model

344

also showed that more than 70% of 3-MCPD dipalmitate was metabolized to 3-MCPD.10, 14 In

345

the present study, except free 3-MCPD, various 3-MCPD fatty acid esters and endogenous

346

substance conjugation metabolites were detected, with possible urine and fecal excretions.

347

In summary, the present study showed that 3-MCPD dipalmitate could be absorbed and

348

distributed to organs and tissues including brain and testis. It could also be metabolized to other

349

3-MCPD diesters, 3-MCPD monoesters, glycidyl ester, 3-MCPD or endogenous substance

350

conjugation metabolites, as well as excreted through both faeces and urine.

351 352

Abbreviations

353

3-MCPD, 3-monochloropropane 1,2-diol; ADME, distribution, metabolism and excretion;

354

AUC0-∞, total area under the plasma concentration-time curve; BW, body weight; CL, clearance

355

rate; Cmax, peak plasma concentration; CNS, central nervous system; DHPMA,

356

2,3-dihydroxypropyl mercapturic acid; LC-MS, Liquid chromatography mass spectroscopy; 19



357

LOD, limit of detection; LOQ, limit of quantification; MRM, multiple reaction monitoring;

358

MRT, mean residence time; NMR, nuclear magnetic resonance; QTOF-MS, quadrupole time of

359

flight-mass spectroscopy; RSD, relative standard deviation; SD, Sprague Dawley; SFC,

360

supercritical fluid chromatography; T1/2, elimination half-life; Tmax, time to reach Cmax; UPCC,

361

Ultra Performance Convergence Chromatography; UPLC, ultraperformance liquid

362

chromatography; TQS-MS, triple quadrupole-mass spectroscopy; Vd, volume of distribution

363 364 365

Funding We gratefully acknowledge the partial financial support from the National Key Research

366

and Development Program of China (Grant No. 2017YFC1600500); the Chinese National

367

Natural Science Foundation (Grant 31501479); and a grant from the Beijing Advanced

368

Innovation Center for Food Nutrition and Human Health, Beijing Technology & Business

369

University (BTBU).

370 371

Supporting Information.

372

MS spectra of standard compounds and identified metabolites, and representative

373

chromatograms of typical metabolites.

374

Supplementary Figure S1. MS/MS spectra of synthetic standard compounds. (A) 3-MCPD

375

dipalmitate, (B) 3-MCPD 1-palmitate, and (C) 3-MCPD 2-palmitate.

20


Page 20 of 37

Page 21 of 37


376

Supplementary Figure S2. MRM chromatogram of 3-MCPD 1-palmitate and 3-MCPD

377

2-palmitate by UPCC-QQQ-MS. (A) Standard of 3-MCPD 1-palmitate (> 90%) and 3-MCPD

378

2-palmitate, (B) Standard of 3-MCPD 2-palmitate (> 90%), (C) Rat intestine sample after oral

379

administration of 1600 mg/kgBW 3-MCPD dipalmitate, (D) Rat liver sample after oral

380

administration of 1600 mg/kgBW 3-MCPD dipalmitate.

381

Supplementary Figure S3. The MS1 spectrum of the metabolites. (A) M0, (B) M2, (C) M3, (D)

382

M4, (E) M5, (F) M6, (G) M7, (H) M8, (I) M9, (J) M10.

383

Supplementary Figure S4. The MS1 spectrum of the metabolites. (A) M11, (B) M12, (C) M13,

384

(D) M14, (E) M15, (F) M16.

385

Supplementary Figure S5. The MS2 spectrum of the parent and the metabolites at a collision

386

energy 20-35 eV. (A) M0, (B) M2, (C) M3, (D) M4, (E) M5, (F) M6, (G) M7, (H) M8, (I) M9, (J)

387

M10.

388

Supplementary Figure S6. The MS2 spectrum of the metabolites at a collision energy 20-35 eV.

389

(A) M11, (B) M12, (C) M13, (D) M14, (E) M15, (F) M16.

390

Supplementary Figure S7. (A) The extract ion chromatogram (EIC) of M17 in a rat liver

391

sample extract, and (B) the MS spectrum of M17. M17 was identified by using a modified DGF

392

GC-MS method.26

393

Supplementary Figure S8. Typical UPLC-QTOF-MS extract ion chromatogram (EIC) and its

394

integration of the liver sample for the 3-MCPD dipalmitate 6 h after oral administration of

395

3-MCPD dipalmitate to rats. 21



396

22


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397

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398

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Creutzenberg, O.; Lampen, A. Relative oral bioavailability of 3-MCPD from 3-MCPD fatty

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15. Buhrke, T.; Frenzel, F.; Kuhlmann, J.; Lampen, A. 2-Chloro-1,3-propanediol (2-MCPD) and

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population. Anal. Bioanal. Chem. 2015, 407, 4823-4827.

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17. Kaze, N.; Watanabe, Y.; Sato, H.; Murota, K.; Kotaniguchi, M.; Yamamoto, H.; Inui, H.;

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Kitamura, S. Estimation of the Intestinal Absorption and Metabolism Behaviors of 2- and

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18. Seefelder, W.; Varga, N.; Studer, A.; Williamson, G.; Scanlan, F. P.; Stadler, R. H. Esters of

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3-chloro-1,2-propanediol (3-MCPD) in vegetable oils: significance in the formation of

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3-MCPD. Food Addit. Contam. Part A Chem. Anal. Control Expo. Risk Assess. 2008, 25,

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19. Gao, B.; Liu, M.; Huang, G.; Zhang, Z.; Zhao, Y.; Wang, T. T.; Zhang, Y.; Liu, J.; Yu, L. L.

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Absorption, Distribution, Metabolism and Excretion of 3-MCPD 1-Monopalmitate after Oral

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Administration in Rats. J. Agric. Food Chem. 2017, 65(12), 2609-2614.

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Pharmacol. 2015, 73, 726-731.

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21. Kong, F.; Pang, X.; Zhong, K.; Guo, Z.; Li, X.; Zhong, D; Chen, X. Increased Plasma

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Exposures of Conjugated Metabolites of Morinidazole in Renal Failure Patients: A Critical

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Role of Uremic Toxins. Drug Metab. Dispos. 2017, 45(6), 593-603.

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22. Zelinková, Z.; Novotný, O.; Schůrek, J.; Velísek, J.; Hajslová, J.; Dolezal, M. Occurrence of

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3-MCPD fatty acid esters in human breast milk. Food Addit Contam Part A Chem Anal

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Control Expo Risk Assess. 2008, 25(6), 669-676.

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23. Lee, J. W.; Uchikata, T.; Matsubara, A.; Nakamura, T.; Fukusaki, E.; Bamba, T. Application

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of supercritical fluid chromatography/mass spectrometry to lipid profiling of soybean. J.

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24. Zhou, Q.; Gao, B.; Zhang, X.; Xu, Y.; Shi, H.; Yu, L. Chemical profiling of triacylglycerols

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25. Trotter, P. J.; Storch, J. Fatty acid esterification during differentiation of the human intestinal cell line Caco-2. J Biol Chem. 1993, 268(14), 10017-23.

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26. Fatty-acid-bound 3-chloropropane-1,2-diol (3-MCPD) and 2,3-epoxipropane-1-ol (glycidol).

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Determination in oils and fats by GC/MS (Differential measurement). DGF Standard Method

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27. Edwards, E. M.; Jones, A. R.; Waites, G. M. The entry of alpha-chlorohydrin into body fluids

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of male rats and its effect upon the incorporation of glycerol into lipids. J. Reprod.

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Fertil.1975, 43(2), 225-232.

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28. Matsui, K.; Mishima, M.; Nagai, Y.; Yuzuriha, T.; Yoshimura, T. Absorption, distribution,

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metabolism, and excretion of donepezil (Aricept) after a single oral administration to Rat.

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Drug Metab. Dispos. 1999, 27(12), 1406-1414.

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29. Lee, J. K.; Byun, J. A.; Park, S. H.; Kim, H. S.; Park, J. H.; Eom, J. H.; Oh, H. Y. Evaluation

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of the potential immunotoxicity of 3-monochloro-1,2-propanediol in Balb/c mice. I. Effect on

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antibody forming cell, mitogen-stimulated lymphocyte proliferation, splenic subset, and

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natural killer cell activity. Toxicology. 2004, 204(1), 1-11.

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30. Lee, J. K.; Byun, J. A.; Park, S. H.; Choi, H. J.; Kim, H. S.; Oh, H. Y. Evaluation of the

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potential immunotoxicity of 3-monochloro-1,2-propanediol in Balb/c mice II. Effect on

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thymic subset, delayed-type hypersensitivity, mixed-lymphocyte reaction, and peritoneal

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macrophage activity. Toxicology. 2005, 211(3), 187-196.

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31. Byun, J. A.; Ryu, M. H.; Lee, J. K. The immunomodulatory effects of

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3-monochloro-1,2-propanediol on murine splenocyte and peritoneal macrophage function in

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vitro. Toxicol. In Vitro. 2006, 20(3), 272-278.

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32. IARC (International Agency for Research on Cancer) Some Chemicals Present in Industrial 27



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and Consumer Products. IARC Monographs on the Evaluation of Carcinogenic Risks to

498

Humans, vol. 101, pp. 349–374 Lyon, France, 2012.

499

28


Page 28 of 37

Page 29 of 37


500

FIGURE CAPTIONS

501

Figure 1. Chemical structures of metabolites.

502

Figure 2. Typical UPLC-QTOF-MS extract ion chromatogram (EIC) of (A) reference standard

503

and (B) sample for the M1 after oral administration of 3-MCPD dipalmitate to rats.

504

Figure 3. MSE spectra of the M1. (A) MS1 spectrum and (B) MS2 spectrum at a collision energy

505

20-35 eV.

506

Figure 4. Possible metabolic pathway and distribution of the metabolites of 3-MCPD dipalmitate

507

in rats (Tissue or body fluid was identified by UPLC-QTOF (Phenyl Column), and the

508

distribution of the metabolites of 3-MCPD dipalmitate in rat based on the peak area. L, Liver; K,

509

Kidney; B, Brain; P, Plasma; Te, Testis; Th, Thymus; I, Intestine; S, Spleen; F, Faeces; and U,

510

Urine; Urine sample was identified by UPLC-QTOF (HILIC Column); 3-MCPD was identified

511

by using a modified DGF GC-MS method.)

29



Page 30 of 37

Table 1. The Kinetic Parameters of 3-MCPD Dipalmitate in Rats After Oral Gavage. Rats ID Unit

1

2

3

4

5

6

Mean

SD

Cmax a

ng/mL

120.00

122.00

124.00

152.00

137.00

155.00

135.00

15.54

Tmax

h

2.50

2.50

2.50

2.50

2.50

2.50

2.50

0.00

h

3.31

3.17

3.21

4.29

5.45

3.76

3.87

0.89

h

4.95

4.76

4.75

5.04

5.55

5.45

5.08

0.34

L/h/g

3.58

3.63

3.65

3.51

3.45

3.17

3.50

0.18

L/g

17.06

16.63

16.90

21.73

27.09

28.65

21.34

5.42

h ng/mL

444.01

440.49

441.14

455.98

464.15

505.02

458.47

24.65

t1/2

b

c

MRT CL

e

Vd

f

d

AUC 0-∞ a

g

Cmax is the maximum concentration of 3-MCPD dipalmitate in rats’ plasma; b Tmax is the time of reach the Cmax after oral administration to rats; c t1/2 is the time

that the concentration of 3-MCPD dipalmitate reduced to half of Cmax; d MRT is the abbreviation of mean residence time, indicate the average time of 3-MCPD dipalmitate stays in the body; e CL is clearance rate of 3-MCPD dipalmitate in rats’ plasma, f Vd is volume of distribution; g AUC is the abbreviation of area under the curve, represent the total amount of 3-MCPD dipalmitate stay in the body.


Page 31 of 37


Table 2. Identification of 3-MCPD Dipalmitate Metabolites in Rat Tissues and Body Fluid. Description M0

Precursor ion [M+Na]

Rt

Molecular

calculated mass

Measured mass

Error

Mass fragment of

(min)

formula

(m/z)

(m/z)

(ppm)

metabolites

9.60

C35H67O4Cl

609.4627

609.4628

0.4

239.2375 551.5025 573.4868

M1

Substitution of C16H30O [M+Na]

8.49

C19H37O3Cl

371.2329

371.2302

-0.2

313.2730

M2

Substitution of C16H31OCl [M+H]/[ M+Na]

8.44

C19H36O3

313.2743/335.2562

313.2733/335.2566

-2.9/1.2

73.0305 95.0697 239.2340

M3

Substitution of C16H30O with C16H28O

9.56

C35H65O4Cl

607.4469

607.4468

-0.2

[M+Na] M4


571.4701 9.81

C37H71O4Cl

637.4939

637.4931

-1.3

[M+Na] M5

Substitution of C16H30O with C18H32O Substitution of C16H30O with C18H30O

9.72

C37H69O4Cl

635.4782

635.4774

-5.0


239.2349 577.5190 599.5034

9.64

C37H67O4Cl

633.4626

633.4626

0

[M+Na] M7

239.2349 579.5331 601.5213

[M+Na] M6

239.2348 549.4868

239.2348 575.5033 597.4858

9.68

C39H67O4Cl

657.4626

657.4626

0

[M+Na]

239.2347 599.5034 621.4859

M8

M4 substitution of C16H30O [M+Na]

8.60

C21H41O3Cl

399.2642

399.2638

-1.0

341.2982

M9


8.52

C21H39O3Cl

397.2485

397.2482

-0.5

339.2817

M10


8.41

C21H37O3Cl

395.2329

395.2327

-0.5

337.2733

M11


8.43

C23H37O4Cl

419.2329

419.2320

-2.1

361.3286

M12

Substitution of C16H30O with glucuronide

6.45

C25H45ClO9

525.2830

525.2843

0.4

331.2244 489.3151

8.04

C29H53N3O9S

620.3581

620.3595

2.3

313.2741 489.3024

conjugation [M+H] M13

S-Glutathione conjugation M2 [M+H]

545.3233 M14

Substitution of C19H35O2Cl [M-H]

8.15

C16H32O2

255.2324 31


255.2322

0.4

239.2319


M15

Substitution of Chlorine with two sulfate

5.00

C35H68O12S2

745.4230

745.4201

Page 32 of 37

-1.5

conjugation [M+H]

605.2513

M16

M12 substitution of C16H30O

M17

3-MCPD

a Urine

sample was analyzed using a UPLC-QTOF (HILIC Column).

b

b

351.1799 409.2241

[M-H]a

3.55

C9H15ClO8

285.0377

285.0389

4.2

177.0384 193.0324

11.90

C3H7ClO2

196

196

--

91 147

3-MCPD was identified using a modified DGF GC-MS method.26

32


Page 33 of 37


O O

O

O OH

O

Cl

O

O

Cl

Cl O

O

O

O O

O

O

O Cl O

M0

Cl

O

O

O

O

O

M4

O

M5

M9

O M11

M10

O

O

O

O

O

HO

Cl

S OH

O

O O

M8

HN

OH

Cl

Cl O

O

O

OH OH

Cl O

O

Cl

Cl OH OH

Cl O

O

(SO4)2

NH2

H N

O

COOH O

O

HOOC M13

M12

O

OH

HO

OH

OH

O

OH

M14

Cl

OH HO Cl

O O

Cl OH

M7

O

O

OH

O

M6

O

Cl

O

Cl O

O

O

OH

O

Cl

O

OH

O O

O O

O O

O

O

Cl O

OH

O

O

Cl O

Cl

O

O

O

HO

O

Cl

Cl O O

O

O

O

M3

O

O

O

O O

HO

M2

M1

O

M16

M17

Figure 1.


M15


Figure 2.

34


Page 34 of 37

Page 35 of 37


Figure 3.

35



Figure 4.

36


Page 36 of 37

Page 37 of 37


TOC Graphic O

O

O

O

O

Cl

Cl

O

O

OH Cl

O

O

O

O O

O

O

O

Cl

O

M0

O Cl

Cl

O O

O

O

O

O

O

O

OH

M12

M11

O

HO

OH HO

O

NH2

H N O

O O O

O

O

O

Cl

HO HO

Cl O M10

M9

OH

Cl OH OH

O

O

S

O

O

Cl O

O

M8

O

Cl OH OH

Cl O

M7

O

O

Cl OH OH

HN

O

O M6

O

O

O

O

Cl

O M5

O

OH Cl

Cl

O

OH

OH O

O

Cl O

Cl

O

O O

O

M4

O

O

O Cl

Cl O

O

M3

O

O

O

O O

M2

M1

O

O

O

O Cl O

(SO4)2 O

COOH O

Cl

O OH

OH OH

O

Cl HO

OH

O

O

HOOC M13

M14

M15

M16

M17

37


Toxicokinetics and metabolism of 3-monochloropropane 1,2-diol (3

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