Development Of Mass Spectrometric Detection Methods For The Identification Of Furan Fatty Acids And Their Influences On Physiological Functions Of Ob/ob Mice

As special fatty acid species containing five-membered furan heterocycles,furan fatty acids are mainly found in plants,algae,and fish.Currently,it is reported that a metabolite of furan fatty acids,3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid?CMPF?,elevated significantly in the plasma of humans with gestational diabetes mellitus,as well as impaired glucose-tolerant and T2D patients.These elevated CMPF could significantly impair the secretion of insulin granule.However,the metabolic pathways of furan fatty acids to CMPF remain elusive.Also,there are very limited evidences reporting the biological functions of furan fatty acids in diabetic subjects.It has been documented that the contents of furan fatty acids in food and biological samples are extremely low.Common detection methods are unfeasible regarding the precise qualification and accurate quantification of furan fatty acids.Therefore,sensitive mass spectrometric detection methods of furan fatty acids in food matrices and biological samples serve as a potential way to unveil the metabolism of furan fatty acids.Additionally,a study of the physiological functions of furan fatty acids in diabetic patients would contribute to a better understanding of the biological relevance of furan fatty acids.In the present study,three modern mass spectrometry approaches including GC-TQ/MS,UPLC/ESI-MS/MS and multidimensional mass spectrometry-based shotgun lipidomics?MDMS-SL?were applied for the qualification and quantification of furan fatty acids in common edible oils,human plasm and natural rubber latices used in food industry.Moreover,the effects of furan fatty acids on the physiological functions of ob/ob mice were investigated.The potential metabolites were quantitatively analyzed.The results are shown as follows:1. Detection and identification of furan fatty acids by mass spectrometryFirstly,GC-TQ/MS was employed to develop a targeted method for qualitative and quantitative analysis of furan fatty acids in common edible oils.In particular,the applicability of several widely used methylation methods?e.g.H₂SO₄-Me OH,BF₃-Me OH,CH₃ONa-CH₃OH and ethereal diazomethane?was investigated.It was demonstrated that H₂SO₄-Me OH and BF₃-Me OH could lead to degradation of the furan ring.Fortunately,a derivatization method involving CH₃ONa-CH₃OH and ethereal diazomethane could largely avoid the above side effects.A targeted quantification method by GC-TQ/MS was developed based on the multiple reaction monitoring?MRM?mode,with a lower limit of quantitation?LLOQ?of 2.0 fmol.The species and contents of furan fatty acids in some common edible oils were analyzed qualitatively and quantitatively.It was demonstrated that furan fatty acids are ubiquitous in marine fish oil.Specifically,a total content of 4.16 mg/g was determined in tuna oil.Secondly,UPLC/ESI-MS/MS was employed to develop a more sensitive method for analysis of furan fatty acids in human plasma.The quantification could not be achieved in the negative mode due to an LLOQ of 50 ng/m L.Furthermore,different derivation reagents,including 4-phenyl-1,2,4-triazoline-3,5-dione,N-[4-?aminomethyl?phenyl]pyridinium,2-bromo-1-methylpyridinium iodide and 3-carbinol-1-methylpyridinium iodide,were used for the sensitivity enhancement.However,an effective derivatization reaction of furan fatty acids could not be observed using4-phenyl-1,2,4-triazoline-3,5-dione.Additionally,severe oxidations were found with respect to the derivatization reaction of furan fatty acids with N-[4-?aminomethyl?phenyl]pyridinium.Fortunately,no side reactions were found using 2-bromo-1-methylpyridinium iodide and 3-carbinol-1-methylpyridinium iodide.An LLOQ of0.05 ng/m L was achieved using this charge-reverse derivatization based on the MRM mode.The detection sensitivity was generally 1000-fold higher than in the negative mode.The targeted quantitation of furan fatty acids in human plasma demonstrated that the concentrations of 3,4-dimethyl-5-propyl-2-furanundecanoic acid?11D3?and 3,4-dimethyl-5-pentyl2-furanundecanoic acid?11D5?in healthy female subjects were 16.24±10.28 ng/m L and 123.83±43.65 ng/m L,respectively.The concentrations of 11D3 and 11D5 in healthy male cohorts were 10.62±3.36 ng/m L and 94.40±27.87 ng/m L,respectively.The precursor ion scan showed that no more furan fatty acid analogues were identified in the plasma of Chinese healthy subjects.Thirdly,multi-dimensional mass spectrometry-based shotgun lipidomics?MDMS-SL?was employed to qualitatively and quantitatively analyze the lipidome of furan fatty acids in three natural rubber latices?i.e.Chicco,Nuk and Suavinex?used in food industries.Results showed that TAG was the most abundant lipid class,in which the highest level of 9M5?6999.45nmol/mg?was found in Suavinex.In addition,two analogues including 3,4-dimethyl-5-propyl-2-furannonanoic acid?9D3?and 11D3 were identified.Furthermore,the qualitative accuracy of furan fatty acid species by MDMS-SL was investigated by GC-MS and UPLC/ESI-HRMS/MS.The close or similar molecular weights of 9M3 and linoleic acid,11D3 and 9D5could not be recognized by MDMS-SL accurately.GC-MS and UPLC/ESI-HRMS/MS results showed that the furan fatty acid analogues including 9M3 and 9D5 were also identified in three natural rubber latices.In addition,11D3 identified by MDMS-SL should be designated as 9D5.2. The metabolism and biological functions of furan fatty acids in ob/ob miceThe metabolism and biological functions of furan fatty acids in ob/ob mice were studied.It was demonstrated that 11D3 could aggravate liver steatosis in ob/ob mice.11D3contributed to significantly increased levels of serum triglycerides?TG?,decreased levels of high-density lipoprotein cholesterol?HDL-C??p<0.05?and increased levels of serum creatinine.The ratio of aspartate transaminase to alanine transaminase,levels of insulin and blood urea nitrogen did not change significantly?p>0.05?.In addition,H&E staining sections showed that ob/ob mice gavaged with 11D3 had acute injuries in renal tubules.With respect to the ob/ob mice gavaged with 11D5,in addition to the significantly increased concentration of creatinine?p<0.05?and mild tubular impairment,other detrimental effects were not observed.The targeted metabolites profiling of furan fatty acids demonstrated that the plasma CMPF levels increased significantly?p<0.05?in ob/ob mice gavaged with 11D3,while the levels of plasma CMPentyl F and the adduct of glucosiduronide and CMPentyl F elevated significantly?p<0.05?in ob/ob mice gavaged with 11D5.These results might suggest that 11D3 and 11D5 serve as the potential metabolic precursors of CMPF and CMPentyl F,respectively.Overall,this study developed sensitive quantification methods by modern mass spectrometry for the identification of furan fatty acids.The applicability of commonly used derivatization methods was analyzed in detail.Additionally,the metabolism and biological functions of furan fatty acids in ob/ob mice were investigated preliminarily.