| Objective: Diabetes mellitus, particularly type 2 diabete is one of the common focuses of attention at home and abroad in recent years. Insulin resistance (IR) is the basic characteristic of type 2 diabetes and is also a key cause of type 2 diabetes risk factors. In 2001 American Diabetes Association (ADA) conference, some scholars pointed out that and type 2 diabete is related to abnormal lipid metabolism more than to abnormal glucose metabolism. In type 2 diabete, abnormal lipid metabolism is a cause of abnormal glucose metabolism, is the primary pathophysiological changes of diabetes and its complications. So, assay of fatty acid profiles in tissues is important for study diabetes .In the state of insulin resistance and diabetes, sugar metabolism is disturbed, the fat mobilization is enhanced, fatty acid oxidation becomes the main source of energy in the body. There are two metabolic pathways for fatty acids oxidation in the cells: one is to be oxidized completely through the mitochondrionβ-oxidation, which is responsible mainly for oxidizing medium and short chain fatty acid and long chain fatty acids (LCFA); the second is to beoxidized through peroxisomalβoxidation, which is responsible mainly for oxidizing very long chain (VLCFA), polyunsaturated (PUFA, including n-3 PUFA and n-6PUFA) and some long chain fatty acids. These two pathways maintain the dynamic balance of fatty acids in the cell. Any wrong with these pathways will result in an accumulation of corresponding fatty acids and then leed to insulin resistance and diabetes through lipotoxity . It will be more toxic to cells when the very long chain fatty acids oxidized in peroxisomer are accumulated in cells.At present, it was found that the levels of total serum free fatty acid are significantly higher in diabetes than in healthy persons . Of this total, the levels of saturated fatty acid increase, the ratio of saturated fatty acids like C16:0,C18:0 to total fatty acid levels decrase. There are also a significantly rise in free fatty acid levels in serum, liver, cardiac muscle, brain and kidney tissues of diabetic rats. However, it was uncertain that in these tissues above, what kinds of fatty acid increased ? which fatty acids are potential for the formation and development of insulin resistance and diabetes? whether do they belong to very long chain fatty acids oxidized in peroxisomer ? In order to answer these questions, we designed and done present study.To assay the changes in fatty acids profile, the common tool is gas chromatography. Because of the poor thermal stability of fatty acids, fatty acids in the sample have to be esterified first and then extracted before chromatography.There is no better determination over a variety of longer than C16 saturated and unsaturated fatty acids extraction and determination at present, therefore, the beginning of the experiment, the method must be established first.For these purposes, the gas chromatography conditions for assaying saturated and unsaturated fatty acids with C16-C26 long chain was identified first, optimized methods to methylate fatty acids from serum, liver, cardiac muscle, brain and kidney tissues, and to extract methyl ester. In this way determination and analysis the changes in saturated and unsaturated fatty acids with C16-C26 long chain profiles in the tissues of high fat diet-induced insulin resistant and high fat diet plus STZ-induced 2 type diabetes SD rats. The relationship between free fatty acids and formation and development of insulin resistance and diabetes was discussed.Methods:1 the establishment of a gas chromatographic method for fatty acid profile Selected polarity column: Agilent DB-WAX polyethylene glycol capillary column (30 m×0.53 mm×1.00μm), the traditional single-stage heating process improvement to three platform temperature program, in the first and second temperature order, add a 190℃temperature platform for 58min of the plateau.Fatty acid methyl ester standard mixture were measured for the improved method,to observe the improved method for a variety of C16-C26 saturated and unsaturated fatty acid methyl ester separation,and determination of retention time, standard curve, the lowest detection limit and recovery rate.2 Optimization of samples free fatty acids methylated and extraction Use SD rats's liver tissue to compare and determine the esterification reagent, and the best number of extraction after esterification ,use the optimization methods to determination relative standard deviation.3 Use optimize method to determine the optimal amount of serum and tissue.4 serum and tissue's fatty acid determinationUse optimize method to calculate fatty acid content of normal group ,insulin resistance group , diabetic group SD rats's serum and liver, cardiac muscle, brain and kidney tissue. 5 Statistical analysisData were expressed as mean±SD. SPSS13.0 soft ware was used. Statistical comparisons were made by T test. A value of P<0.05 was considered significant.Results:1 the establishment of a gas chromatographic method for fatty acid profile1.1 Determination of chromatographic conditionsChromatographic conditions were eventually identified as:GLC(Agilent 7890A)using a 30 m×0.53 mm×1.00μm polyethylene glycol column (DB-WAX, Agilent). The column oven was programmed to(1) rise after 1 min at 150–190℃at 10℃/min(2)rise after 58 min at 190-230℃(3)keep 230℃15min with a Nitrogen gas flow rate of 80 cm/s as the carrier gas.Improved chromatographic conditions can separat C16: 0, C16: 1, C18: 0, C18: 1, C18: 2, C18: 3, C20: 0, C20: 4, C20: 5, C22: 0, C22: 4 , C22: 5, C22: 6, C24: 0, C26: 0 fatty acid methyl ester standard, separation is good. 1.2 Determine the retention timeFatty acid methyl esters standard retention time: C16:0 7.104±0.012 min, C16:1 7.522±0.027 min, C18:0 11.023±0.009 min, C18:1 11.594±0.020 min, C18:2 13.024±0.012 min, C18:3 15.390±0.008 min, C20:0 18.300±0.022 min, C20:4 25.385±0.014 min, C20:5 30.647±0.009 min, C22:0 32.232±0.014 min, C22:4 45.697±0.017 min, C22:5 55.726±0.022 min, C24:0 58.611±0.010 min, C22:6 60.815±0.018 min, C26:0 71.426±0.025 min.1.3 calibration curve: C16:0 Y=1587.9X+2690(R~2=0.9997), C16:1 Y=1704.4X+48.867(R~2=0.9998), C18:0 Y=1731.8X+1041.6(R~2=0.9997), C18:1 Y=1719.5X+3260.7(R~2=0.9997), C18:2 Y=3194.0X-1262.8(R~2=0.9990), C18:3 Y=3326.2X+32.191(R~2=0.9997), C20:0 Y=4978.2X-199.33(R~2=0.9974), C20:4 Y=1787.6X-742.95(R~2=0.9991), C20:5 Y=2057.8X-43.33(R~2=0.9990), C22:0 Y=1939.3X-47.97(R~2=0.9988), C22:4 Y=1200.5X+210.13(R~2=0.9998), C22:5 Y=3006.3X+290.76(R~2=0.9998), C24:0 Y=1783.3X+45.32(R~2=0.9993), C22:6 Y=2673.1X-358.81(R~2=0.9992), C26:0 Y=2192.9X+95.102(R~2=0.9985). X:Concentration of fatty acid methyl esters(μg/μl),Y:Peak area 1.4 detection limit: C16:0 0.0783μg/ml, C16:1 0.0730μg/ml, C18:0 0.0718μg/ml, C18:1 0.0723μg/ml, C18:2 0.0390μg/ml, C18:3 0.0374μg/ml, C20:0 0.0250μg/ml, C20:4 0.0696μg/ml, C20:5 0.0605μg/ml, C22:0 0.0641μg/ml, C22:4 0.1036μg/ml, C22:5 0.0414μg/ml, C24:0 0.0698μg/ml, C22:6 0.0465μg/ml, C26:0 0.0567μg/ml.1.5 recovery amplitude:95.2% -107.5% .2 Optimization of samples free fatty acids methylated and extractionIn esterification reagents, acetyl chloride's catalytic efficiency is the best; the best times for extraction is three,relative standard deviation is 1.94% -8.62% and 2.33% -11.60%.3 Determine the optimal dosage of serum and tissuesSerum, liver, cardiac muscle, brain and kidney tissues's optimal dosage: 800μl, 800mg, 800mg, 65mg, 500mg.4 The changes of insulin resistance and diabetic rats serum and liver tissues,cardiac muscle, brain and kidney tissues's free fatty acids4.1 Compared with the normal group, the fatty acids's changes in insulin resistance rats:In Serum, total fatty acid, SFA, UFA, SFA / UFA, n-3PUFA, n-3PUFA / n-6PUFA, LCFA, VLCFA were significantly higher, n-6PUFA were significantly lower, the differences were statistically significant. In liver,SFA/UFA,n-3PUFA/ n-6PUFA were significantly higher, n-3PUFA,n-6PUFA were significantly lower,the differences were statistically significant.In heart,total fatty acid,SFA,UFA,SFA/UFA,LCFA were significantly higher,the differences were statistically significant.In brain,n-3PUFA,VLCFA were significantly higher,the differences were statistically significant.In kidney,total fatty acid,SFA,UFA,SFA/UFA,LCFA were significantly higher, n-3PUFA were significantly higher,the differences were statistically significant.4.2 Compared with the normal group, the fatty acids's changes in diabetic rats:In Serum, total fatty acid, SFA, UFA, SFA / UFA, n-3PUFA, LCFA, VLCFA were significantly higher, n-6PUFA were significantly lower, the differences were statistically significant.In liver,SFA/UFA, n-3PUFA/ n-6PUFA,VLCFAwere significantly higher,UFA,n-6PUFA were significantly lower,the differences were statistically significant.In heart,total fatty acid, SFA,UFA,n-6PUFA,LCFA were significantly higher, SFA/UFA,n-3PUFA,n-3PUFA/ n-6PUFA,VLCFA were significantly lower, the differences were statistically significant.In brain,SFA/UFA were significantly higher,total fatty acid,UFA,n-3PUFA,n-6PUFA,n-3PUFA/n-6PUFA,LCFA,VLCFA were significantly lower,the differences were statistically significant.In kidney,total fatty acid,SFA,UFA,SFA/UFA,LCFA were significantly higher, n-3PUFA, n-6PUFA,n-3PUFA/n-6PUFA,VLCFA were significantly low,the differences were statistically significant.4.3 Compared with the insulin resistance group, the fatty acids's changes in diabetic rats:In Serum,n-6PUFA were significantly higher,SFA/UFA, n-3PUFA,n-3PUFA/ n-6PUFA,VLCFA were significantly lower,the differences were statistically significant. In liver,n-3PUFA,n-3PUFA/ n-6PUFA,VLCFA were significantly higher,the differences were statistically significant.In heart,total fatty acid,UFA,n-6PUFA ,LCFA were significantly higher, SFA/UFA,n-3PUFA,n-3PUFA/ n-6PUFA,VLCFA were significantly lower,the differences were statistically significant.In brain,SFA/UFA were significantly higher,UFA,n-3PUFA, n-3PUFA/n-6PUFA were significantly lower,the differences were statistically significant.In kidney,n-3PUFA,n-6PUFA,n-3PUFA/ n-6PUFA,VLCFA were significantly lower,the differences were statistically significant.Conclusion:1 Established the method by gas chromatography for fatty acids C16-C26, repeatability and accuracy are good, and is a useful tool for routine testing C16-C26 fatty acids.2 In Insulin resistance and diabetes status, the increased long chain and very long chain fatty acid levels may involved in increased total fatty acids of. serum3 In insulin resistance and diabetes status, there are different changes in fatty acid profiles in liver, heart , brain and kidney of rats.In Liver, the increase in very long chain fatty acids may involved in the increase of the total fatty acid content. Of these, C22: 6(DHA) was increased significantly;In heart, the increase in long chain fatty acids may involved in the increase in total fatty acid content. Of these, C22: 6 was significantly reduced;In Brain, decreased changes in saturated and unsaturated fatty acids with C16-C26 long chain dose not involved in the increase of the total fatty acid content . Of these, C22: 6 were significantly reduced;In Kidney tissue, levels of long-chain fatty acids were increased significantly, C22:6 were significantly reduced.
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