| PurposeThis experiment is divided into two parts. In the first part,2HiO was used as a tracer to mark the plasma albumin in rats, to establish a detection method of alanine marked by 2H2O, which is the basis for the study of protein synthesis rate. In the second part, rats were fed with low Lysine diet (0.2%), normal Lysine diet (0.9%) and high Lysine diet (1.6%) for a long time, we studied the metabolic kinetics of albumin and the rate of serum albumin synthesis, analysis the sensitivity of albumin metabolic kinetics index to lysine, to lay a good foundation for further study on Lysine metabolic kinetics experiment in human.Methods1.2H2O was used as a tracer to inject into rats, and then take the rats’blood and separate the plasma.2. Plasma albumin was purified by the method of TCA/Ethanol, and the free amino acid residues were removed.3. The purified albumin was identified by SDS-PAGE as albumin.4. Albumin hydrolysis by acid, and then collect alanine after hydrolysis. At the same time, we collected blood free alanine.5. Separation and purification of alanine from two sources:albumin hydrolysis or blood.6. Derivative of alanine and detected by GC/MS, we determined the feasibility of detection method of alanine marked by 2H2O.1. Design and produce three kinds of feed by AIN-93G, respectively low Lysine diet (0.2%), normal Lysine diet (0.9%) and high Lysine diet (1.6%). We detected the contents of diet ingredients to ensure the diet standard.2.72 male SD rats (12 weeks old, weight about 320g) were randomly divided into three groups, each group had 24 rats. They were intake with low Lysine diet (0.2%), normal Lysine diet (0.9%) and high Lysine diet (1.6%) for 8 weeks freely.3. Since 9 weeks, every rats were mark blood free alanine and blood protein with 2H2O, until 18 weeks end. And record rats’ weight and food intake on the last day of every week between the 18 weeks.4. Rats were sacrificed in batches at 8,9,11,13,14 and 18 week. Blood was collected to detect the abundance of 2H-alanine in albumin and the abundance of 2H-alanine in plasma free alanine by GC/MS. Before detect the abundance of 2H-alanine, the amount of albumin was detected by ALB kit, and then purification and identification of it by TCA/Ethanol.5. The three groups rats were respectively drawn diagrams about 2H tracer change trend in albumin, calculating the equation of albumin metabolic kinetics and the rate of albumin synthesis, and then compare the results of the three groups, analysis the effect of different Lysine level on albumin metabolism dynamics.ResultsWe can detect the abundance of alanine derivatives groups (molecular weight 144 and 159) marked by 2H2O in albumin in 90 minutes after 2H2O was used as a tracer to inject into rats, and the abundance of the group in three groups was consistent, it means that 2H2O can successfully mark albumin, which is in accordance with the needs of the requirement of experiment.1. Diet composition test showed that lysine content of the three kinds of diet were:0.23g/100g, 0.89g/100g,1.61 g/100g, according with the requirement of experiment.2. Food intake of the low Lysine diet (0.2%) group were significantly lower than the other two groups. Although there was no difference in food intake between normal Lysine diet (0.9%) group and high Lysine diet (1.6%) group.3. Body weight of the three groups have significant difference, the weight from light to heavy order were:low Lysine diet (0.2%) group, normal Lysine diet (0.9%) group, high Lysine diet (1.6%) group.4. Serum albumin amount of the high Lysine diet (1.6%) group was significantly higher than the other two groups. And there was no diffidence between normal Lysine diet (0.9%) group and low Lysine diet (0.2%) group.5. The rate of serum albumin synthesis of low Lysine diet (0.2%) group, normal Lysine diet (0.9%) group and high Lysine diet (1.6%) group respectively were 13.8%,15.3%,35.5%.6. The kinetic equation of albumin synthesis of the three groups were as follows:low Lysine diet (0.2%) group normal Lysine diet (0.9%) group high Lysine diet (1.6%) groupConsults1. We can detect alanine marked by 2H2O in albumin in 90 minutes after 2H2O was used as a tracer to inject into rats, it means that 2H2O can successfully mark albumin, which is in accordance with the needs of the requirement of experiment.2. In 18 weeks experimental period, compare with the rate of serum albumin synthesis o f normal Lysine diet (0.9%) group rats, low Lysine diet (0.2%) can decrease the rate sli ghtly, and high Lysine diet (1.6%) can increase the rate of serum albumin synthesis obvi ously. Rate of serum albumin synthesis and metabolic kinetics equation is sensitive to highly sine intake status, while is not sensitive to low Lysine intake status. |
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