Synthesis,detection And Acute Toxicity Of 2-MCPD Esters

Chloropropanol esters have been detected in food such as refined vegetable oils,baking and fried food.Chloropropanol esters have potential reproductive toxicity,nephrotoxicity and immunotoxicity,and are a pollutant produced in the process of food thermal processing.The homologue 2-chloropropanol(2-MCPD)ester has not received sufficient attention relative to the 3-chloropropanol(3-MCPD)ester.The reason is that the toxicological data 2-MCPD esters is lacking.On the other hand,synthesis process of 2-MCPD esters are complicated,with too much impurities and difficult to purify,this is tricky for researchers.The purpose of this thesis was to design a new synthetic route,using diethyl malonate as raw material to synthesize free 2-MCPD under mild conditions,and then obtain two different structures of 2-MCPD monoester and diester by esterification.And the structure were characterized by mass spectrometry,nuclear magnetic resonance spectroscopy,infrared spectroscopy and circular dichroism.The method of detecting 2-MCPD monoester by ultra performance convergence chromatography tandem time-of-flight mass spectrometry(UPCC-q TOF)was improved,and the production of P-2-MCPD was confirmed by the palmitate mode reaction.The acute toxicity of 2-MCPD palmitic acid monoester(P-2-MCPD)and 2-MCPD palmitic acid diester(PP-2-MCPD)to Swiss mice was determined by the maximum limit method.The results showed that the LD50 of P-2-MCPD and PP-2-MCPD were more than 5000 mg/kg body weight(BW),and the acute toxicity was comprehensive evaluated by changes in food intake and body weight,histopathological,and blood biochemical parameters.Using the mouse Leydig cell line TM3 as a model,the reproductive toxicity of six 2-MCPD esters was studied based on cell viability.Further,Western blotting was used to determine the effects of P-2-MCPD and PP-2-MCPD on expression of main functional proteins in testosterone synthesis and apoptosis and necrosis pathways,including steroid hormone acute regulatory proteins(StAR),Bax,Bcl-2,Cleaved caspase 3,MLKL and RIP xp.This paper lays the foundation for further exploration of the formation mechanism and toxicity mechanism of 2-MCPD esters.The main results are as follows:1.Using diethyl malonate as starting material,chlorination of 1,3-Dichloro-5,5-dimethylhydantoin to obtain diethyl chloromalonate(DECM)under acid catalysis;then DECM was reduced to free 2-MCPD by sodium borohydride in cold methanol solution;the fatty acid and free 2-MCPD were esterified under DMAP and DCC catalysis by Steglich esterification.Structural characterization was carried out by mass spectrometry,nuclear magnetic resonance spectroscopy,infrared spectroscopy and circular dichroism.The overall yield of 2-MCPD monoesters and diesters were about 50-54%and 56-59%.2.Improved UPCC-q TOF analysis method to separate 2/3-MCPD mono esters was optimized by column,modifier mobile phase,automatic back pressure regulator(ABPR)pressure,column temperature and elution gradient.The optimum conditions for the addition of the modifier were Torus 2-PIC column(2-picolylamine);modifier mobile phase was methanol;ABPR 1800 psi;column temperature 50?;flow rate 1 mL/min;gradient elution:0.5-10%methanol.The method had good separation efficiency for different 2-MCPD monoester or 3-MCPD monoester in 12 mins;and can also separate different 2/3-MCPD monoesters of the same fatty acid.For palmitic acid,the ratio of 3-/2-MCPD monoester formed in monoglyceride,glycidyl ester and triglyceride is 2.9-4.5:1;under the same molar conditions,glycerol diester is more likely to form 2/3-MCPD monoester.3.The acute toxicity of P-2-MCPD and PP-2-MCPD to Swiss mice was determined by the maximum limit method.The results showed that none of the Swiss mice died after 14 days of administration of 2-MCPD ester.The LD50 of P-2-MCPD and PP-2-MCPD were greater than 5000 mg/kg BW.According to the classification criteria of GHS,both P-2-MCPD and PP-2-MCPD should be classified as Nontoxic compounds.Four Swiss mice were observed with vertical hair and diarrhea in a short time;compared with the control,the average body weight change and average food intake of Swiss mice after P-2-MCPD and PP-2-MCPD were no significant difference within 14 days;there was no statistical difference in organ morphology and weight,urea,creatinine,alanine aminotransferase,aspartate aminotransferase.The P-2-MCPD group decreased content of serum creatine kinase and PP-2-MCPD group rised the levels of a-hydroxybutyrate dehydrogenase in Swiss mice,and the serum testosterone levels in Swiss mice of P-2-MCPD and PP-2-MCPD groups were significantly reduced.Histopathological analysis found that high doses of P-2-MCPD and PP-2-MCPD(5000 mg/kg BW)caused partial renal tubular necrosis,partial constructive cells distribution disorder and shedding,ley dig cells decreased in several mice as compared with the control group.While no significant changes were observed in the heart,liver,spleen,lung,epididymis,and thymus.4.The cytotoxicity of six 2-MCPD esters on mouse Ley dig cells TM3 was determined by MTT assay.It was found that except for OO-2-MCPD,there were dose-response relationships among the other five 2-MCPD esters.The cytotoxicity of P-2-MCPD,PP-2-MCPD,O-2-MCPD and S-2-MCPD on TM3 was determined by LDH method.The results showed that P-2-MCPD was a significant increase the LDH release between 40-320?M and there was a dose-response relationship.The effect of P-2-MCPD and PP-2-MCPD on the expression of several key proteins in testosterone synthesis,apoptosis and necrosis pathway was determined by Western blotting.It was found that only the expression level of Cleaved caspase 3 was significantly reduced between 6 h and 24 h after administration and the time response relationship was evident.The results showed that 2-MCPD ester has certain cytotoxicity to mouse Leydig cells TM3,especially P-2-MCPD and PP-2-MCPD,and the mechanism of P-2-MCPD may be related to Cleaved caspase 3 protein which guided by apoptosis.