重水标记法测定日粮赖氨酸摄入量对大鼠骨骼肌蛋白代谢动力学的影响

发布时间：2018-02-11 07:51

本文关键词： 赖氨酸骨骼肌蛋白重水示踪剂分数合成率　出处：《扬州大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:给予大鼠一定浓度的~2H_2O作为示踪剂标记动物体内骨骼肌蛋白,以期建立氘标记丙氨酸的检测方法,为研究骨骼肌蛋白分数代谢率提供方法保证;在日常饮食状态下,分别观察大鼠在较长时间内摄入含有低(0.2%)、正常(0.9%)、高(1.6%)剂量的赖氨酸饲料,骨骼肌蛋白分数合成率是否相同,分析骨骼肌蛋白代谢动力学指标是否对赖氨酸摄入量敏感,骨骼肌蛋白代谢动力学是否一致。分析~2H_2O标记法是否能有效测定骨骼肌蛋白的合成动力学参数,是否适用于长期标记实验,从而为进一步进行人体赖氨酸动力学实验研究奠定基础。方法:第一部分:(1)~2H_2O标记大鼠身体水90min,收集血液;~2H_2O标记大鼠骨骼肌一周,收集四肢肌肉,提取骨骼肌蛋白和血浆游离氨基酸。将身体水中的2H与丙酮发生交换,萃取丙酮。(2)酸水解法水解骨骼肌蛋白,收集水解后丙氨酸,同时收集血液游离丙氨酸。(3)对骨骼肌水解后丙氨酸经0.22μm微孔膜对水解液进行过滤,对血浆游离丙氨酸进行分离和纯化。(4)对丙氨酸进行衍生化处理,身体水的萃取物经过微孔膜过滤,分别经GC/MS检测,确定了氘标记丙氨酸检测方法的可行性。第二部分:(1)参考AIN-93G各氨基酸成分比例设计三种饲料赖氨酸含量分别为低(0.2%)、正常(0.9%)、高(1.6%)。(2)健康成年雄性Sprague-Dawley(SD)大鼠72只,12周龄,体重320g左右,随机分为3组,每组24只。分别给予低、正常和高剂量赖氨酸饲料,喂养8周,自由进食,正常饮水。(3)自第9周开始,给予大鼠~2H_2O以标记血液游离丙氨酸、骨骼肌蛋白以及身体水,标记至第18周结束。18周内每周称量大鼠体重并记录每组大鼠的进食量。(4)在8,9,11,13,14,18周处死大鼠,收集血液和四肢肌肉,作为样本,用凯氏定氮法测骨骼肌蛋白的含量,GC/MS检测骨骼肌蛋白中的2H-丙氨酸的丰度、血浆游离丙氨酸中2H的丰度及身体水的2H丰度,观察各丰度的变化情况。(5)分别绘制各组大鼠骨骼肌蛋白中2H示踪剂的变化趋势图,计算2H标记骨骼肌蛋白的代谢动力学方程和蛋白质的合成率,比较不同日粮赖氨酸组所得结果的异同,观察身体水的2H丰度的变化情况,分析不同赖氨酸摄入水平对机体骨骼肌蛋白代谢动力学具有怎样的影响。结果:第一部分:(1)腹腔注射~2H_2O90min后,身体水中丙酮的分子量为58/59,血浆游离丙氨酸衍生物的分子量为99/100、158/159基团的丰度均可测出,确定了身体水中2H丰度出峰时间为2.3min左右。(2)大鼠骨骼肌蛋白中丙氨酸衍生物的分子量为99/100、158/159基团的丰度均可测出,通过与标准库对比,确定骨骼肌丙氨酸衍生物的出峰时间为8.45min左右,且结果具有一致性。第二部分:(1)大鼠饲料的检测结果,低、正常和高剂量饲料中赖氨酸含量分别为0.23g/100g、0.89g/100g和1.61g/100g,均符合实验要求。(2)三组大鼠的日均进食量分别为21.64g、23.07g和23.78g,低剂量赖氨酸组大鼠的进食量明显减少,而正常剂量组与高剂量赖氨酸组大鼠的进食量并未受到显著影响。(3)高剂量赖氨酸组与正常剂量组大鼠体重均值分别是低剂量组大鼠体重均值的1.20倍和1.17倍,三组大鼠体重均值具有显著性差异,随赖氨酸摄入量增高,体重呈增加趋势。(4)低、正常和高剂量组大鼠身体水中2H丰度均值分别为:0.1116、0.1190和0.1180,大鼠身体水被氘标记,摄入~2H_2O后2H能够在很短时间内与全身各组织的水达到平衡并且稳定。(5)三组大鼠的骨骼肌蛋白含量分别为18.28%、18.87%和18.77%,无显著性差别。(6)低、正常和高剂量赖氨酸组的骨骼肌蛋白分数合成率分别为11.6%,12.8%和18.8%。(7)骨骼肌蛋白合成动力学方程为:低剂量赖氨酸组ft=0.1604×(1-e-0.116t);正常剂量赖氨酸组ft=0.158×(1-e-0.128t);高剂量赖氨酸组ft= 0.1464×(1—e-0.1878t)。结论:腹腔注射~2H_2O 90min后,即能检测出血浆游丙氨酸和身体水被氘标记,说明摄入~2H_2O后能够成功的标记血浆游丙氨酸并且2H能在很短时间内与全身各组织的水达到平衡;每日给予4%~2H_2O作为日常饮水,持续1周后,即能检测出骨骼肌蛋白中的丙氨酸被氘标记,说明该方法能够成功标记骨骼肌蛋白。与正常剂量赖氨酸(0.9%)摄入量组大鼠骨骼肌蛋白分数合成率相比,低剂量赖氨酸(0.2%)摄入量组大鼠骨骼肌蛋白分数合成率略有降低,高剂量赖氨酸(1.6%)摄入量组提高了大鼠骨骼肌蛋白的分数合成率。从骨骼肌蛋白分数合成率、合成动力学方程及曲线图可知,骨骼肌蛋白的分数合成率和代谢动力学方程能敏感反映机体的高赖氨酸摄入状态,但对低赖氨酸摄入状态不敏感。
[Abstract]:Objective: rats were given a certain concentration of ~2H_2O as tracer marker in animal skeletal muscle protein, in order to establish the detection method of deuterium labeled alanine, provide guarantee for the research of skeletal muscle protein metabolism scores; in the diet condition, rats were observed for a long time with low intake (0.2%), normal (0.9%) (1.6%), high lysine feed dosage, skeletal muscle protein fractional synthesis rate is the same, the analysis of skeletal muscle protein metabolism kinetics index of lysine intake sensitive skeletal muscle protein metabolism kinetics are consistent. Analysis of synthetic kinetic parameters of ~2H_2O labeling method can effectively determine whether skeletal muscle protein, is suitable for long term labeling experiments, which provides a basis for further study of human kinetics experiment of ammonia acid lysine. Methods: the first part: (1) ~2H_2O labeling of rat body water 90min, blood collection; ~2H_2O Rat skeletal muscle markers for a week, collecting the muscles of the limbs, extraction of skeletal muscle protein and plasma free amino acid. The exchange, 2H and acetone water extraction of acetone body. (2) acid hydrolysis of skeletal muscle protein, collected after hydrolysis of alanine, and collected blood free alanine. (3) of skeletal muscle after hydrolysis of alanine 0.22 mu of M microporous membrane of hydrolysate was filtered on plasma free alanine was isolated and purified. (4) derivatization of alanine, the body of water extract by microporous membrane filtration, were detected by GC/MS, to determine the feasibility of deuterium labeled alanine detection method. The second part: (1 the amino acid composition of the proportion of AIN-93G) reference design of three kinds of dietary lysine levels were low (0.2%), normal (0.9%), high (1.6%). (2) adult male Sprague-Dawley (SD) 72 rats, aged 12 weeks, weighing about 320G, were randomly divided into 3 groups, each group of 2 4. Were given low, normal and high dose of lysine amino acid feed, feeding for 8 weeks, free eating, normal drinking water. (3) since the beginning of the ninth week, the rats were given ~2H_2O with marker free blood alanine, skeletal muscle protein and water body, to mark the end of the eighteenth week.18 weeks weighing the weight of rats and the food intake of rats in each group were recorded. (4) at 8,9,11,13,14,18 weeks the rats were sacrificed, blood was collected and the muscles of the limbs, as samples, the content measurement of skeletal muscle protein nitrogen by the Kjeldahl method, the abundance of skeletal muscle protein was detected in GC/MS 2H- alanine, 2H abundance and abundance of plasma free amino acid C water body in 2H, to observe the changes in the abundance. (5) were drawing trend of each rat skeletal muscle protein 2H tracer kinetics equation, and the protein synthesis rate 2H markers of skeletal muscle protein, comparison of different dietary lysine group results The similarities and differences, changes in 2H abundance observed in body water, analysis of different lysine intake level of what is the effect of skeletal muscle protein metabolism kinetics. Results: the first part: (1) after intraperitoneal injection of ~2H_2O90min, the molecular weight of the body in water acetone was 58/59, the molecular weight of plasma free phenylalanine derivatives as abundance can be 99/100158/159 the group measured, determine the body water abundance of 2H peak time is about 2.3min. (2) the molecular weight of alanine derivatives in rat skeletal muscle protein in the 99/100158/159 group can be abundance determined by comparing with the standard library, determine the skeletal muscle alanine derivatives peak time is about 8.45min, and the results are consistent. The second part: (1) rat feed test results, low, normal and high dose dietary lysine content were 0.23g/100g, 0.89g/100g and 1.61g/100g, are in line with the experimental requirements. (2) of the three groups of rats daily food intake were 21.64g, 23.07g and 23.78g, lysine intake of low dose group rats significantly reduced food intake and normal dose group and high dose group of lysine in rats was not affected. (3) the average weight of high dose lysine amino acid the group with normal dose group rats were 1.20 times the average weight of low dose group rats and 1.17 times, the average weight of rats in the three groups have significant difference, with the increase of amino acid intake, body weight increased. (4) low, normal and high dose of body water rats 2H abundance respectively: 0.1116,0.1190 and 0.1180 rats, body water is deuterium labeled, after intake of ~2H_2O 2H can in a very short period of time and body tissues of water balance and stability. (5) protein in skeletal muscles of rats in the three groups were 18.28%, 18.87% and 18.77%, no significant difference between the low (6). Normal, and high dose of Lai Skeletal muscle protein fractional synthesis rate of ammonia acid group were 11.6%, 12.8% and 18.8%. (7) protein synthesis in skeletal muscle dynamics equation: low dose of lysine was ft=0.1604 * (1-e-0.116t); the normal dose of lysine was ft=0.158 * (1-e-0.128t); high dose lysine group (1 - 0.1464 x ft= e-0.1878t). Conclusion: intraperitoneal injection of ~2H_2O 90min, which can detect the plasma alanine and swim body of water by deuterium labeling, that after intake of ~2H_2O successfully mark plasma alanine and 2H can swim and body tissues of water balance in a very short period of time; as a daily dose of 4%~2H_2O daily drinking water, after 1 weeks that is, can detect protein in skeletal muscle by deuterium labeled alanine, indicating successful markers of skeletal muscle protein. The method with the normal dose of lysine (0.9%) intake group rats skeletal muscle protein fractional synthesis rate compared to the low dose of lysine intake (0.2%) The amount of skeletal muscle of rats protein fractional synthesis rate decreased slightly, the high dose of lysine intake group (1.6%) increased the fractional synthesis rate of skeletal muscle protein in rats. The skeletal muscle protein fractional synthesis rate, synthesis kinetics equation and the curve graph shows that the fractional synthesis rate and Metabolic Kinetic Equation of skeletal muscle protein can be sensitive reflect the high lysine intake of the body, but for the low lysine intake is not sensitive.

【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R151

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