Synthesis of 2-Monochloropanol Fatty Acid Esters and Their Acute Oral Toxicities in Swiss Mice

Article abstract of DOI:10.1021/acs.jafc.9b01083

A novel synthetic route was designed, developed, and utilized to synthesize six high-purity 2-monochloropropanediol fatty acid esters (2-MCPD esters), a group of potential processing-induced food contaminants. A chlorine atom was introduced to C-2 of a diethyl malonate molecule, which was reduced by NaBH₄ and followed by esterification using fatty acids. The reaction products were isolated and purified using silica gel columns to obtain three 2-MCPD monoesters and three diesters at about 50-54% and 56-59% yields, respectively. In addition, 2-MCPD monopalmitate and dipalmitate were examined for their acute oral toxicities in Swiss mice. The LD₅₀ values of 2-MCPD mono- and dipalmitate were greater than 5000 mg/kg body weight (BW), along with detectable nephrotoxicity and testicular toxicity. The results of this study may promote future investigation of MCPD ester toxicology and detection.

Full text of DOI:10.1021/acs.jafc.9b01083

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Food Safety and Toxicology
Synthesis of 2-monochloropanol fatty acid esters,
and their acute oral toxicities in Swiss mice
Zhongfei Zhang, Puyu Yang, Boyan Gao, Guoren Huang, Man Liu, and Liangli (Lucy) Yu
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b01083 • Publication Date (Web): 15 Mar 2019
Downloaded from http://pubs.acs.org on March 17, 2019
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Journal of Agricultural and Food Chemistry
Synthesis of 2-monochloropanol fatty acid esters, and their acute oral
toxicities in Swiss mice
Zhongfei Zhang,1 Puyu Yang,1 Boyan Gao,1,2,3* Guoren Huang,1 Man Liu,1,4 and
Liangli Lucy Yu3*
1Institute of Food and Nutraceutical Science, School of Agriculture & Biology,
Shanghai Jiao Tong University, Shanghai 200240, China
2Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing
Technology & Business University (BTBU), Beijing 100048, China
3Department of Nutrition and Food Science, University of Maryland, 0112 Skinner
Building, College Park, Maryland 20742, United States
4College of Life Science, Jiangsu Normal University, Xuzhou 221116, China
Contact Information of the Corresponding Authors: Boyan Gao, Tel: 86-21-
3420-4538, Email: gaoboyan@sjtu.edu.cn; Liangli (Lucy) Yu, Ph.D. Tel: 301-0405-
0761, Fax: 301-314-3313, Email: lyu5@umd.edu
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ABSTRACT
A novel synthetic route was designed and developed, and utilized to synthesize
six high purity 2-monochloropropanediol fatty acid esters (2-MCPD esters), a group
of potential processing-induced food contaminants. A chlorine atom was introduced
to C-2 of a diethyl malonate molecule, which was reduced by NaBH4 and followed by
esterification using fatty acids. The reaction products were isolated and purified using
silica gel columns to obtain three 2-MCPD monoesters and three diesters at about
50%-54% and 56%-59% yields, respectively. In addition, 2-MCPD monopalmitate
and dipalmitate were examined for their acute oral toxicities in Swiss mice. The LD50
values of 2-MCPD mono- and di-palmitate were greater than 5000 mg/kg body
weight (BW), along with detectable nephrotoxicity and testicular toxicity. The results
of this study may promote future investigation of MCPD ester toxicology and
detection.
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KEYWORDS: 2-MCPD esters, synthesis, diethyl malonate, acute oral toxicity
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INTRODUCTION
Fatty acid esters of chloropropanol are a group of contaminants potentially
formed during food thermal processing. Four groups of chloropropanol esters have
been reported, including 3-monochloropropane-1,2-diol (3-MCPD) esters, 2-
monochloropropane-1,3-diol (2-MCPD) esters, 1,3-dichloropropane-2-ol (1,3-DCP)
esters and 1,2-dichloropropane-3-ol (1,2-DCP) esters1. Chloropropanol esters were
detected in many food categories, including hydrolyzed vegetable proteins (HVP)2,
refined vegetable oils3, 4, baked cereal products5, infant formula6, and human breast
milk7, 8. 3-MCPD esters have been reported at the greatest content among the four
chloropropanol esters9, and have been mostly researched in the past decades for their
detection, formation mechanism, acute toxicity, metabolisms and mitigation9.
Recently, 2-MCPD esters have been detected in refined olive oil, vegetable oil,
soy sauce and other processed foods8-10 because of the advancement of analytical
methods. For instance, Yan and others 10 reported a concentration range of 0-6 mg/kg
for 2-MCPD esters in olive and other vegetable oils, whereas 3.5 and 9 μg/kg of 2-
MCPD esters was detected in the crude and deodorized kenaf seed oils, respectively11.
Braeuning and colleagues treated Wistar rats with 10 and 53 mg/kg BW of 2-MCPD
and 2-MCPD dipalmitate every day for 28 days, and the toxic effects were observed
on the transcriptomic responses in rat kidney, liver and testis12, whereas the proteomic
effects were found in rat heart 13 and testes14. The acute toxicity of free 2-MCPD has
been reported by Jones15 and ESAF report9, and the results indicated that the LD50
values of free 2-MCPD were about 260 mg/kg BW in male rats or 50-60 mg/kg BW
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in rats determined using the UP and Down method. Buhrke and colleagues16 reported
a conversion of 2-MCPD esters to free 2-MCPD in cultured Caco-2 cells, suggesting
the potential toxicity of 2-MCPD esters through their free 2-MCPD metabolite. These
findings warrant additional research to investigate the toxicity of 2-MCPD ester
isomers, and their presence and distribution in food and biological samples. The
primary limiting factor for conducting such studies is the availability of high purity 2-
MCPD ester isomers.
Three synthetic routes have been reported for preparing 2-MCPD fatty acid
esters17-19. Two of the routes used allyl alcohol17 or glycidol18 as a starting material to
synthesize 2-MCPD intermediate. The problems of these two methods are low yields
of 2-MCPD ester and the co-presence of 3-MCPD esters. The third method used 1,3-
bibenzyloxy-propanol to synthesize 2-MCPD esters with a greater purity19. But this
method had a yield less than 28%, and involved a hydrogenation step, which creates
safety concerns and requests special facility. New methods for high purity 2-MCPD
ester synthesis are needed for their detection and toxicity investigations.
In this study, a new synthetic route was developed and used to prepare six high-
purity 2-MCPD mono- and diesters with greater yields under mild experimental
conditions. After the synthesis, the acute toxicities of 2-MCPD 1-palmitate (P-2-
MCPD) and 2-MCPD 1,3-dipalmitate (PP-2-MCPD) were investigated using Swiss
mice. This is the first investigation of LD50 values for 2-MCPD mono- and diesters in
mice. The results of this study may promote future investigation of MCPD ester
toxicology and detection.
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MATERIAL AND METHODS
Materials. Diethyl malonate was purchased from Shanghai Maclean Biochemical
Co., Ltd. (Shanghai, China); diethyl chloromalonate, 1,3-dichloro-5,5-
dimethylhydantoin (DCDMH) purchased from Adamas Reagent Co., Ltd. (Shanghai,
China); heptafluorobutyryl imidazole, sodium borohydride,
dicyclohexylcarbodiimide, 4-dimethyl aminopyridine were supplied by Aladdin
Reagent Co., Ltd. (Shanghai, China); palmitic acid, stearic acid were purchased from
TCI (Shanghai, China) Chemical Industry Development Co., Ltd.; oleic acid was
from Acros organics (Morris Plains, NJ, USA).Deuterated methanol and deuterated
chloroform were spurchased from CIL (Andover, MA, USA); sulfuric acid, sodium
hydrogen hydride, sodium chloride, methanol, dichloromethane, tert-butyl methyl
ether (MTBE) and calcium hydride were purchased from Sinopharm Chemical
Reagent Co., Ltd. (Shanghai, China); silica gel (Merck 60), 2-propanol and methanol
(LC-MS grade) were obtained from Merck (Darmstadt, Germany); the water used in
the laboratory was supplied by Millipore pure water meter (Darmstadt, German).
High purity nitrogen (99.999%) was purchased from Rui Li chemical gas Co., Ltd
(Shanghai, China). GHP membrane (0.2 µm) was purchased from Pall Cooperation
(NYC, USA).
Synthesis of 2-MCPD esters. A new synthetic route was designed to synthesize
2-MCPD esters through chlorination, reduction and esterification reactions (Figure 1).
Chlorination reaction was refined from a previous publication20. Briefly, 3 mmol of
sulfuric acid was added dropwise into a mixture of 10 mmol diethyl malonate, 7.5
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mmol DCDMH and 15 mL acetonitrile. The reaction was carried out with stirring at
70 °C for 2 h. After cooling to ambient temperature, the solvent in the filtered solution
was evaporated, and solid residue was re-dissolved in 15 mL dichloromethane. After
washing with distilled water three times, the organic phase was dried with anhydrous
sodium sulfate overnight. After filtration, the dichloromethane was evaporated under
a reduced pressure to give a colorless oily liquid, which is the intermediate compound
diethyl chloromalonate (DECM) (Figure 1, step A). The structure and purity of
DECM were confirmed by 1H NMR and MS analyses.
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DECM was reduced using sodium borohydride according to a previous
publication with modification21. Briefly, 1 mL of 1 M sodium borohydride solution in
methanol was slowly added into 1 mmol DECM in 5 mL methanol kept in an ice bath.
The reaction mixture was warmed up to ambient temperature and reacted until no
DECM was detected by thin-layer chromatograph (TLC) examination. After the
reaction was completed, 1 mL of saturated ammonium chloride solution was added,
followed by filtration. Methanol was removed under a reduced pressure, and the crude
free 2-MCPD was purified by silica gel column chromatography with a mobile phase
containing n-hexane/ethyl acetate/methanol (45/45/10, v/v/v) (Figure 1, step B).
Esterification reaction was modified from a protocol reported by Steglich and
colleagues22. Briefly, 2 mmol 2-MCPD, 5 mmol fatty acid and 15 mg of DMAP was
mixed in 10 mL anhydrous dichloromethane, and the mixture was kept in an ice bath.
Then 5.1 mmol of DCC in 10 mL of dichloromethane was added to the reaction
mixture. The reaction temperature was raised to ambient temperature and kept
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overnight. After filtration, the liquid was collected and evaporated to dryness under a
reduced pressure, washed with dichloromethane to ensure removal of 1,3-
dicyclohexylurea. After added an appropriate amount of dichloromethane, the
solution was washed sequentially with an equal volume of 0.5 M hydrochloric acid,
saturated NaHCO3 and distilled water, and dried over anhydrous sodium sulfate. The
crude 2-MCPD diesters were separated and purified using a 200 mesh silica gel
column guided by TLC.
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For the synthesis of 2-MCPD monoesters: 1 mmol of 2-MCPD, 2.1 mmol of
DCC and 15 mg of DMAP were dissolved in 5 mL of the dried dichloromethane. One
mmol of fatty acid in 5 mL dichloromethane was added in the mixture. Other reaction
conditions and crude 2-MCPD ester isolation and purification conditions were the
same as described above (Figure 1, step C).
Characterization
GC-MS and UPCC-Q TOF MS. A modified GC-MS method was utilized based
on a previously reported ptotocol23. Briefly, 10 μL free 2-MCPD or 3-MCPD was
dissolved in 2 mL of n-hexane, 40 μL of heptafluorobutyrylimidazole was added and
sealed, vortexed for 1 min and incubated for 20 min at 70 °C. After cooling to
ambient temperature, 2 mL of deionized water was added, and vortexed for 1
minutem. The supernatant solution was collected and dried by anhydrous sodium
sulfate, and analyzed by GC-MS (Agilent 7890A-5975C, USA).
UPCC-Q TOF MS method was modified from the protocol by Jumaah and
colleagues24. Briefly, Waters UPCC system with Waters Torus 2-PIC column (3.0
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mm ×100 mm, 1.7 μm); column temperature, 50 °C; ABPR, 1800 psi; mobile phase A
and B were high pure CO2 and methanol, respectively; gradient elute: 0-3 min, 100%
A; 3-8 min, 99% A; 8-9 min, 97% A; 9-10 min, 100% A; 10-12 min, 100% A;
makeup solvent was methanol with 0.1 mM ammonium formate, flow rate was 0.4
mL/min. Waters Q TOF MS system, resolution mode, ESI source, positive; capillary
3.0 kV; sampling cone, 30.0 V; source temperature 120 °C; desolvation temperature
450 °C; desolvation gas flow, 700 L/Hr.
NMR. 1H-NMR and 13C-NMR were recorded using a Bruker AVANCE III 600
MHz spectrometer (Rheinstetten, Germany). The solvents of 2-MCPD and its esters
were methanol-d4 and chloroform-d1, respectively. Tetramethylsilane (TMS) was
used to determine zero point of chemical shift, and all the other chemical shifts of
proton and carbon were referenced to TMS.
IR. 2-MCPD and 2-MCPD esters were directly detected using a Thermo Fisher
Nicolet 6700 FT-IR (Madison, USA) with ATR (Ge crystal).
CD spectrum. Samples were dissolved in methanol, and detected on a Jasco J-
815 CD spectrometer (Tokyo, Japan). The blank was methanol.
Animal study. A 14-day acute oral toxicity study was performed to evaluate the
acute toxic effects of 2-MCPD 1-palmitate and 2-MCPD 1,3-dipalmitate in Swiss
mice25. The experimental protocol was approved by the Institutional Animal Care and
Use Committee in Shanghai Jiao Tong University (experiment ID: A2015024).
Briefly, 30 Swiss mice (15 females and 15 males, 3-4 weeks old) were received from
Shanghai SLAC laboratory animal Co. Ltd (Shanghai, China). All the mice were
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acclimatized for 5-7 days. Since preliminary experiment showed no death at the
dosages of 1000 mg/kg BW, 2000 mg/kg BW and 5000 mg/kg BW, a limit dose
method was chosen to assess the acute toxicity of P-2-MCPD and PP-2-MCPD
according to the Organization for Economic Cooperation and Development
guideline26. The Swiss mice were randomly divided into three groups (5 male and 5
female mice in each group). Group 1 was the vehicle control group and orally
administrated with extra virgin olive oil; group 2 and 3 were orally administrated
5000 mg/kg BW of P-2-MCPD and PP-2-MCPD in the extra virgin olive oil,
respectively. 4 hours later, water was free accessible to all animals. The body weight
and food intake were recorded every day for 14 days. During the period, the behaviors
of mice, including eyes, fur, mucous membranes, secretions, excretions and
autonomic activity, were carefully observed every day.
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Serum biochemistry and histological analysis. On the 14th day, blood samples
were collected from the orbital sinus after the carbon dioxide anesthesia. The serum
samples were prepared by centrifuging blood samples at 3500 rpm for 10 minutes at 4
C, and serum was transferred to a new 1.5 mL centrifuge tube and stored at -80 C
before analysis. Testosterone, α-hydroxybutyrate dehydrogenase (HBDH), serum
creatine kinase (CK), alanine aminotransferase (ALT), aspartate aminotransferase
(AST), urea nitrogen and creatinine in serum were measured by King Med
Diagnostics, Inc. (Shanghai, China).
After blood samples were collected, mice were sacrificed by cervical dislocation
followed by a careful gross necropsy. Heart, liver, spleen, lung, kidney, thymus, brain
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and testis were collected and weighed. Parts of the organs were fixed with 4%
paraformaldehyde overnight at 4 C , while testis and epididymis were fixed in
modified Bouin’s solution overnight at 4 C .The rest parts of the organs were
temporarily kept under liquid nitrogen, and transferred to a -80 C refrigerator before
sample preparation. Sections with Hematoxylin-eosin (HE) staining were produced by
the Shanghai Medical College Basic Medicine Public Technology Platform. Briefly,
the fixed tissue were dehydrated, embedded in paraffin, and sectioned (5 μm). All
tissue sections were deparaffinized by xylene, dehydrated by gradient ethanol, and
then stained with HE. Histopathological changes were record by an Olympus
microscope (Tokyo, Japan).
Statistical analysis. Data were expressed as the mean ± standard deviation (SD)
for each experiment. Differences between means were determined by the analysis of
one-way analysis of variance (ANOVA) and Tukey’s test, using the Statistical
analysis system (SAS) software (V 9.2, SAS INSTITUTE INC, North Carolina,
United States). Statistical significance was declared at P < 0.05.
RESULTS AND DISCUSSION
Synthesis. A new synthetic route for high purity 2-MCPD mono- and diesters
was designed and developed for the first time with diethyl malonate as the starting
compound (Figure 1). A chlorine atom was introduced to C-2 using DCDMH to
obtain diethyl chloromalonate (Figure 1, step A). Diethyl chloromalonate was reduced
to free 2-MCPD with a purity greater than 98% using sodium borohydride (Figure 1,
step B). The free 2-MCPD was esterified with free fatty acids using a Steglich
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esterification reaction (Figure 1, step C). The most critical and innovative step of this
synthetic route was the reduction reaction by sodium borohydride in methanol to
obtain high purity free 2-MCPD without a high temperature condition. This reduction
reaction (step B) was mild and safer compared to the palladium catalyzed
hydrogenation reaction19. The synthesized 2-MCPD had no detectable 3-MCPD
impurity as examined by GC-MS analysis after derivatization with heptafluorobutyryl
imidazolium (Figure S1A and S1B). The purity of 2-MCPD was 98% by NMR
analysis (Figure S1C). The yield of the reduction reaction was about 86%.
The chlorinating reaction (step A) was optimized in this study27. Two
chlorinating reagents, N-chlorosuccinimide (NCS) and 1,3-dichloro-5,5-
dimethylhydantoin (dichlorohydantoin, DCDMH) were evaluated, and DCDMH was
selected as the chlorinating reagent because of its lower toxicity and greater reaction
efficiency. Besides, three catalysts including silica gel, ammonium chloride and
sulfuric acid were compared for their capacity in catalyzing the chlorination
reaction28. Sulfuric acid resulted in a greater yield and required a shorter reaction
time. The solvent for the chlorination reaction was changed to ethanol to reduce
transesterification reaction. The yield of the chlorination reaction in this experiment
was 82% (Figure 1, step A). In addition, diethyl chloropropionate is a more
commercially available starting material.
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While Steglich esterification reaction of free 2-MCPD inevitably produces both
monoesters and diesters, the mono- and diesters of 2-MCPD were separated on a
silica gel column, and mobile phase for 2-MCPD diesters was n-hexane/ethyl acetate
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(10:1, v/v) followed by n-hexane/ethyl acetate (20:1, v/v). The yield of 2-MCPD
diesters was 80-84%. The mobile phase for 2-MCPD monoesters was n-hexane/ethyl
acetate (4:1, v/v), and the yield was 72-77%. The overall yield were 50%-54% and
56%-59% for monoesters and diesters with a same fatty acid, respectively. Compared
with previously reported synthetic approaches, the method developed in this study
synthesizes 2-MCPD esters at a greater yield and purity. A total of three 2-MCPD
monoesters (P-2-MCPD, S-2-MCPD and O-2-MCPD), as well as three 2-MCPD
diesters (PP-2-MCPD, SS-2-MCPD and OO-2-MCPD) were synthesized in the
present study (Figure 1).
Characterization. Six 2-MCPD esters and the critical intermediates were
examined and confirmed for their chemical structures using high resolution MS,
NMR, IR and CD analyses. For instance, the chemical structure of diethyl
chloromalonate (DECM) was determined by MS and NMR analyses. The ion at m/z
217.0242 was the peak of [M+Na]+ (Cal. m/z value 217.0244) (Table S1); the 1H
NMR spectrum showed two equal methyl signals at δ 1.32 (t, J = 7.2 Hz, 6H) and two
equal methylene signals at δ 4.31 (qd, J = 7.1, 1.6 Hz, 4H), the signals of proton [δ
4.86 (s, 1H)] was H-2 at C-2 with chlorine (Table S2); the signals of carbonyl,
methylene and methyl carbons were at δ 164.54, 63.22 and 13.92 in the 13C NMR
spectrum,, respectively, the carbon signal of C-2 was at δ 55.49 (Table S2).
As shown in Figure 2, the BPI spectrum (A), MS1 (B) and MS2 (C) spectra were
used to characterize PP-2-MCPD.The retention time of PP-2-MCPD was 4.31 min,
the ion at m/z 604.5071 was the peak of [M+NH4]+ (Cal. m/z value 604.5072), while
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the m/z 609.4636 (Cal. m/z 609.4626) was [M+Na]+ in MS1 spectra29. Since the
sodium salt was not volatile, the sampling cone needed to be cleaned frequently.
Volatile ammonium formate was selected as the ionization enhancing agent; the ion
with m/z 331.2407 was the peak of [M-palmitic acid]+, while the m/z 551.5059 was
[M-Cl]+ in MS2 spectrum (Figure 2C). The structure of PP-2-MCPD was confirmed
by 1H and 13C NMR spectra as shown in Figure 3: the signals of proton at δ 4.34-4.27
(m, 4H) was two equivalent methylene groups of H-1 and H-3, the signal of proton at
δ 4.24 (p, J = 5.6 Hz, 1H) was the characteristic signal of H-2 of PP-2-MCPD, while
other signals of proton were assigned to the fatty acid19; the characteristic carbon
signal of PP-2-MCPD was at δ 54.88. The NMR, MS and IR data of DECM, 2-
MCPD and the six 2-MCPD esters were shown in Figures S2-S6 and Tables S1-S2.
Results also suggested that the synthesized 2-MCPD esters had a high purity (> 98%,
Figure 3) and could be used for further toxicology studies.
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Acute toxicity. The acute toxicities of P-2-MCPD and PP-2-MCPD were
evaluated using a 14-day Swiss mice model. No significant difference was observed
in the body weight gain (Figure S7A) and food intake (Figure S7B) between the
control, P-2-MCPD and PP-2-MCPD groups in 14 days. It was also noted that several
Swiss mice treated by P-2-MCPD and PP-2-MCPD were inactive and sluggish; some
had piloerection and diarrhea, but improved in 3-4 days compared with the control
mice. After 14 days, no death was observed, and no visually abnormalities in tissues
and organs were observed during dissection in the P-2-MCPD and PP-2-MCPD
groups. The LD50 values of P-2-MCPD and PP-2-MCPD in Swiss mice were above
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5000 mg/kg BW. Both 2-MCPD mono- and dipalmitate can be classified as nontoxic
compounds according to GHS classification system. To our best knowledge, this is
the first report of LD50 values for 2-MCPD esters.
Liu and colleagues reported the LD50 values for P-3-MCPD and PP-3-MCPD at
2676.81 mg/kg BW and over 5000 mg/kg BW in Swiss mice25, respectively. Taking
together, P-2-MCPD may be less toxic than its counterpart P-3-MCPD. These results
agreed to a previous observation that 2-MCPD esters were generally less toxic than
their counterpart 3-MCPD esters9. Other toxic effects of 2-MCPD esters in vivo
remain unclear.
It has been shown that the primary target organs of 3-MCPD esters were kidney
and testis25. Also noted was that free 2-MCPD was toxic to heart, striated muscle,
kidney, liver30 and testis13,14 at oral administration of 10 mg/kg BW per day. In this
study, no significant difference was observed in serum urea nitrogen, creatinine, ALT
or AST among the three treatment groups (Figure S8). Taking into account the
previous observation that P-3-MCPD intake at a dose of 4162 mg/kg BW significantly
increased urea nitrogen and creatinine25, 2-MCPD esters might be less toxic to liver
and kidney than their counterpart 3-MCPD esters. Compared with the control group,
the creatine kinase in the P-2-MCPD group was significantly reduced, but no change
was observed in the PP-2MCPD group. The α-hydroxybutyrate dehydrogenase, a
biomarker of myocardial injury, was significantly increased in the PP-2-MCPD group,
but not in the P-2MCPD group. In addition, P-2-MCPD and PP-2-MCPD significantly
decreased the testosterone level in the mice (Figure S8G).
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No organ damage was observed by histopathological analysis of heart, liver,
spleen, lung, epididymis and thymus, but possible damage was observed for the
kidneys and testis in several mice. P-2-MCPD and PP-2-MCPD at the dose of 5000
mg/kg BW resulted in partial tubule necrosis in kidney cortex of the mice (Figure 4B
and Figure 4C). As showed in Figure 5B and Figure 5C, possible testicular
constructive cell disorder and shedding, and leydig cell reduction were observed in
the male mice exposed to P-2-MCPD and PP-2-MCPD compared with the control.
These observations agree with those documented by Buhrke and colleagues12 that free
2-MCPD and PP-2-MCPD had an toxic effect on the kidneys and testicles. It needs to
be pointed out that the tubule necrosis in kidney was less than that induced by P-3-
MCPD25. P-3-MCPD induced tubular necrosis, focal degeneration and protein casts in
the kidney of dead mice treated with a single oral dose of 2040 mg/kg BW or
greater25. The same renal lesions were also found in the kidneys of dead mice in the
PP-3-MCPD treated group25.
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Both 3-MCPD palmitate25 and free 3-MCPD31 showed significant testicular
toxicity, which could cause desquamation and disorganization of testicular
constructive cells, and reduced spermatids, leydig cells and sperms in the epididymis.
Similar trends could also be observed in the mice of P-2-MCPD and PP-2-MCPD
groups, but sperms did not decrease significantly (data not shown).
In addition, CD spectrum (Figure S9.) had no obvious absorbance in the
wavelength range of 200-600 nm, suggesting that the synthesis and separation under
the present study conditions do not produce a single chirality, and the prepared 2-
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MCPD monoesters were all racemic. The CD spectrum was determined for the 2-
MCPD esters since the optical isomers of 3-MCPD esters differed in their toxic
effects32. Porter and others32 observed that the R- isomer of free 3-MCPD had
hepatotoxicity and nephrotoxicity, but the counterpart S- isomer had reproductive
toxicity. Additional studies are also needed to determine the long-term effects of
consuming optical isomers 2-MCPD and 3-MCPD esters.
In summary, a new route has been designed and developed using diethyl
malonate as the starting material to obtain greater yield and purity 2-MCPD mono-and
diesters. This study also determined the LD50 values of greater than 5000 mg/kg BW
for P-2-MCPD and PP-2-MCPD in Swiss mice for the first time, along with
nephrotoxicity and testicular toxicity using a 14-days limit test study. Both P-2-
MCPD and PP-2-MCPD may be nontoxic compounds according to GHS
classification system. The results suggest the importance of distinguishing 2-MCPD
and 3-MCPD esters in food safety risk assessment. Additional investigations are
needed to compare the toxicity and the molecular mechanisms involved in the toxic
effects of 2- and 3-MCPD esters. Further investigation of acute toxicity of 2-MCPD
esters in different animals and using more isomers is needed to better understand their
acute toxicity and its potential species difference.
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ABBREVIATIONS LIST
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324
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326
327
328
329
330
331
332
333
334
335
2-MCPD, 2-monochloro-1,3-propanediol; 2-/3-MCPD, 2-MCPD and 3-MCPD; P-2-
MCPD, 2-MCPD 1-Palmitate; PP-2-MCPD, 2-MCPD 1,3-Dipalmitate; S-2-MCPD,
2-MCPD 1-Stearate; SS-2-MCPD, 2-MCPD 1,3-Distearate; O-2-MCPD, 2-MCPD 1-
Oleate; OO-2-MCPD, 2-MCPD 1,3-Dioleate; DCC, Dicyclohexylcarbodiimide;
DMAP, 4-dimethylaminopyridine; DCM, dichloromethane; Q-TOF MS, Quadrupole-
time of flight mass spectrometer; FT-IR, Fourier transform infrared; CD speutrum,
Circular binary spectrometer; TM3, TM3 mouse testis leydig cells; MTT, Thiazolyl
Blue Tetrazolium Bromide;
ACKNOWLEDGEMENT
We thank Wilmar (Shanghai) Biotechnology Research & Development Center
for providing palmitic acid.
FUNDING SOURCES
This research was supported by The National Key Research and Development
Program of China (Grant No. 2018YFD0901000, 2017YFC1600500).
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FIGURE CAPTION
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Figure 1. The synthetic route of 2-MCPD esters. DCDMH, 1,3-dichloro-5,5-
dimethylhydantoin; DECM, diethyl chloromalonate;2-MCPD, 2-monochloro-1,3-
propanediol; DCC, Dicyclohexylcarbodiimide; DMAP, 4-dimethylaminopyridine;
DCM, dichloromethane;
Figure 2. The UPCC-Q TOF chromatogram of A) PP-2-MCPD, B) MS1 and C) MS2
spectrums. BPI, base peak intensity.
Figure 3. A) 1H NMR and B) 13C NMR spectrums of PP-2-MCPD.
Figure 4. Representative kidney sectioned and stained with HE (×400) from mice
treated with A) extra virgin olive oil; B) 5000 mg/kg of P-2-MCPD in extra virgin
olive oil; and C) 5000 mg/kg of PP-2-MCPD in extra virgin olive oil. B) and C) show
small part of renal tubular necrosis (indicated by the arrows) in several mice as
compared with A).
Figure 5. Representative testis sectioned and stained with HE (×400) from mice
treated with A) extra virgin olive oil; B) 5000 mg/kg of P-2-MCPD in extra virgin
olive oil; and C) 5000 mg/kg of PP-2-MCPD in extra virgin olive oil. B) and C) show
partial constructive cells distribution disorder and shedding (indicated by the arrows)
in several mice as compared with A).
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O
O
O
O
O
RT, stir
over night
N
N
O
O
Cl
Cl
O
O
Cl
O
DECM
A
DCDMH
NaBH4, MeOH
B
℃
0
,0.5 h; RT for 7h
C
OH
OH
2-MCPD
℃
OR1
OH
OR1 DMAP, DCM, at 0
Cl
Fatty acid
Cl
Cl
DCC, stir
OR1
2-MCPD monoester
2-MCPD diester
R1=palmitic, stearic, oleic acid
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Figure 1.
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Figure 2.
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Figure 3.
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Figure 4.
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Figure 5.
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