Establish A Analysis Method Of Free Fatty Acids’ Content In Serum And Its Relationship With Hyperlipidemia

Background:The direct source of calories in an organism(adipose tissue breaks down neutral fat into free fatty acids(FFAs),which is used as a post-energy source,after the energy required from glycogen for muscle activity is depleted)is FFAs(fast)as well as a product of neutral fat decomposition,also a substance needed to maintain long-term activity,and one of the substances that cause oxidative stress.Most of the FFAs are combined with albumin,presenting in the blood.Therefore,the concentration of endogenous FFAs in the serum is closely related to biological lipid metabolism,sugar metabolism and endocrine function.The level of the lipids in the blood is became higher after abnormal lipid metabolism,then leading to atherosclerosis,coronary heart disease and other diseases about seriously endanger the health of life,all of them belong to hyperlipidemia.therefore,lipid metabolism is closely related to hyperlipidemia.Objective:Establish a biological analysis method of free fatty acids’ content used the gas chromatography-mass spectrometry(GC-MS);Then improve the detected method of GC-MS to find the relationship between serum FFAs content and hyperlipidemia in mice,through comparing the normal group and the positive control group with the model group.Method and contents: 1.Improved of a biological analysis method for the determination of FFAs in mice serum by GC-MS.2.Methodological verification of the analytical method of serum free fatty acid content.3.Detect the content of mice serum FFAs in the normal group,the positive control group and the model group after 28 days,and to explore the relationship between the content of FFAs in the serum of mice,golden hamster and hyperlipidemia.Mainly through improved the sample preparation,chromatographic separation conditions,mass spectrometry detection parameters and so on,as well as built the methodological verification,verified the GC-MS analysis method of the sample has been built.The problem of instability of the target compound is solved by sample derivatization.The sample preparation is based on the most common method of fatty acid determination is hydrolysis and methylation,while methyl esterification can esterify “-COOH” of free fatty acids into “-COCH3”,reducing its polarity reduces and instability,promoting the separation of unsaturated isomers,for all of that it becomes one of the best sample processing methods for measuring serum FFAs.According to 2020 the Pharmacopoeia of People’s Republic of China’s requirements of biological sample analysis methods,in this study,we have investigated the quantitative analysis method of standard addition with the linear range,sensitivity,precision,accuracy,dilution,stability,matrix effect and recovery rate,etc. After that,we also compared the content of FFAs in SPF mice serum with golden hamsters.To explore the relationship between the content of FFAs in the serum of mice,golden hamster and hyperlipidemia.Results and discussion: 1.Improved the method: the injection volume is 2.0μL,the split ratio is 1:1,the solvent delay time is 2.9 min,the ion source voltage is 70 e V,the full scan mode and range are 35-450 m/z,the number of target compounds is adjusted to 26.2.Established and validated a GC-MS analytical method for measuring the concentration of FAMEs in serum successfully.The standard curve has good linearity,with R values is 0.99,had great intra-day and inter-day’s precision(RSD%≤13.99%)and accuracy(relative error:85%-115%),the linear range is 0.10-1000 ng/m L,the matrix effect is small,the sample recovery range is 85.57%-115.18%,the residual effect is small,The matrix effect is small,and the stability is good within four hours at 4℃ and 25℃.3.The content of FFAs in the serum of mice and golden hamsters is closely related to the development of hyperlipidemia.Simvastatin has a significant intervention effect on the content of FFAs in the serum.In this article,the serum samples were processed by methyl ester derivatization to convert free fatty acids into fatty acid methyl esters to increase their stability,so that the fragment ion spectrum can be displayed in the temperament instrument,which provides a reference for future biological sample processing;In this paper,we chose a rapid,sensitive and reliable biological analysis method for the determination of the content of 26 free fatty acids in the serum was established.The content of 26 free fatty acids in the serum of two experimental animals was detected,and the content of various fatty acids was found to be associated with hyperlipidemia.The relationship between hyperlipidemia and rat species was found to be closely related.It was found in mice that simvastatin has a significant callback effect on the content of 13 FFAs in the serum and a significant improvement effect on the content of 1 FFA in the serum.It is an early detection and prognostic evaluation of lipid metabolism-related diseases,especially high The research on lipemia-related diseases provides a certain scientific basis.And in the literature summary,it is found that there is a lack of quantitative analysis of large-scale FFAs species,and there is no literature on comparison of serum FFAs between rat species.Therefore,this article uses experimental research to set foot in this unknown field and provide ideas and directions for research in this area.