稳定核素示踪法应用于内毒素诱导兔高代谢脓毒症的代谢研究

【作者】 杨子昂；

【导师】 吴肇汉；

【作者基本信息】 复旦大学 ， 外科学， 2006， 博士

【摘要】 脓毒血症休克是引起重症监护室病人死亡的重要原因之一。在感染初期，机体往往出现以体温升高、心率加快、血压正常或轻微下降为特点的高动力状态。此时机体的代谢表现为高代谢和分解代谢，因此又称此期为高代谢脓毒症。尽管给予机体足量的热卡和氨基酸，但此时机体的瘦体组织仍大量丢失，延缓了病人的恢复速度并且降低了机体抵抗疾病的能力，甚至可导致更为严重的后果。研究表明：此期的针对性治疗极其重要，如能给予足量且合适的液体复苏措施，并去除病因、炎症介质及有害的代谢物质等，再提供合理的支持治疗，将能阻止病情进展为脓毒症的失代偿期，从而提高治疗效果，降低死亡率。高代谢脓毒症时，机体存在着非常显著的高代谢和分解代谢。糖代谢表现为有氧代谢明显减弱，糖酵解过程加强，而同时糖异生作用也增强，导致血糖、血乳酸升高以及代谢性酸中毒，促进了病情的恶化；蛋白质代谢表现为外周的结构蛋白分解代谢显著加强，氨基酸入血增加，此时肝脏对氨基酸的摄取、转运能力以及氨基酸糖异生作用也明显增强，临床上表现为明显的负氮平衡、瘦体组织丢失以及免疫功能下降；而乳酸作为导致机体代谢性酸中毒的重要因素，其代谢同样出现明显改变，糖酵解过程增强导致其血浓度显著上升，同时它还起着提供能量的作用，并还可通过糖异生作用再生成葡萄糖，是一个综合复杂的过程。因此探索高代谢脓毒症时机体器官间底物的流动过程对于临床营养支持策略是非常重要的。研究机体物质代谢的传统方法有如体重、血常规、血浆蛋白、转铁蛋白的测定以及身体测量等静态营养评价方法，还包括能量代谢以及氮平衡等动态营养评价方法。但它们都只能测定代谢改变的结果，并不能告知我们此时机体各类营养物质流动和代谢的具体参数和途径。稳定核素示踪法作为测定活体内物质代谢的方法，具有精度高、安全、有效的特点，在国外已应用于动物实验和人体实验，但国内目前尚缺乏相应的研究资料。本课题拟通过内毒素持续灌注法建立实验兔的高代谢脓毒症模型，探讨其各类物质的代谢改变，并用¹³C标记的葡萄糖、氨基酸和乳酸作为示踪剂，利用气相质谱仪测定呼出气中的¹³C标记的CO₂含量，利用气相色谱．质谱联用法（GC-MS）测定血中¹³C标记的各类物质含量，探讨高代谢脓毒症时各类营养底物的流动过程。实验分为三部分：研究目的：脓毒症是目前临床上较为棘手的问题，一旦转入休克期，其死亡率极高，因此目前的研究方向主要是避免机体转入失代偿休克期。而在高动力代偿期，由于机体同时存在着极为明显的高代谢状态，导致营养物质利用差、酸碱平衡紊乱以及免疫功能下降等，因此了解此期机体各物质的代谢情况对于治疗脓毒症极为重要。而要加强这方面的研究，首先需要建立完善的高代谢脓毒症动物模型，文献有采用兔、鼠、羊和猪等建立高代谢脓毒症模型的报道，由于兔对内毒素的反应与人类相似，且可获得足够血量，因此本研究拟采用微量的LPS（内毒素）持续静脉输注法建立兔高代谢脓毒症模型，模型建立标准为：实验兔体温显著升高、心率显著加快而血压正常或轻度下降。材料和方法：1)分两组各取健康新西兰兔8只，禁食12小时但可随意饮水；2)采用氯胺酮和安定肌肉注射麻醉后，分别行气管插管、颈动脉、颈静脉及股动脉插管；3)通过颈静脉以10ng／kg·min速度持续灌注LPS（内毒素）3h；4)采用多功能心电监护仪和动态有创血压监护仪监测各组实验兔的体温、心率、呼吸及血压变化；5)取血测定血常规及血浆内毒素含量；6)数据分析处理采用ANOVA分析。结果：LPS持续灌注3h后，结果如下：1)高代谢脓毒症组的心率升高至334.0次／min、体温升高至41.0℃，而对照组则分别为274.9次／min和38.2℃，两组间有显著的统计学差异，p=0.000；血压在高代谢脓毒症组是97.1mmHg，而对照组为95.9mmHg，两组间无统计学差异；脓毒症组的呼吸加快至94.3次／min，对照组则提高至76.4次／min，两组间无统计学差异（p=0.212）；2)其血浆内毒素含量为0.088EU／ml，而对照组为0.013 EU／ml，两组间有显著的统计学差异，p=0.002；3)血中性粒白细胞比值升高至82.73％，对照组则为64.39％，两组间有显著的统计学差异（P=0.001）；白细胞总数则明显下降至1.90×10⁹／ml，而对照组则为7.58×10⁹／ml，p=0.001，两组间有显著的统计学差异；两组间的红细胞数及血细胞比容均无统计学差异。结论：通过内毒素诱导法可成功建立高代谢脓毒症兔模型，表现出心率明显加快、体温明显升高，而血压不变等典型的临床表现。第二部分内毒素诱导兔高代谢脓毒血症的代谢改变研究目的：在高代谢脓毒症期，由于长期处于严重的高分解状态，机体会出现体重明显减轻、血浆白蛋白降低、贫血、机体免疫力下降以及代谢紊乱等难以控制的临床表现，其中代谢紊乱是最为棘手的问题之一，而其还可引起机体的连串反应，进一步加重病情。因此探讨高代谢脓毒症时的代谢紊乱情况对于提高脓毒症的治疗效果非常重要，本研究拟通过前面建立的高代谢脓毒症兔模型，评估高代谢脓毒症实验兔的糖代谢、能量代谢、氨基酸代谢以及酸碱平衡改变。材料和方法：1)分两组各取健康新西兰兔8只，建模方法同第一部分；2)采用强生公司血糖试纸测定实验兔基础血糖值及实验后30min、60min、120min以及180min时的血糖值；3)采用乳酸测试盒测定实验前后的血乳酸含量；3)血样处理后，采用氨基酸分析仪测定建模前后的血浆氨基酸含量，对其变化进行评估；4)采用自动血气分析仪测定实验兔基础的、30min、60min、120min以及180min的动脉血气，评估高代谢脓毒症时的酸碱平衡情况；5)采用能量代谢仪对实验兔的二氧化碳生成量（VCO₂）、氧耗量（VO₂）及能量消耗（EE）以及呼吸商（RQ）进行监测；6)数据分析处理采用ANOVA分析。结果：建模后，高代谢脓毒症组实验兔的代谢改变如下：1)血糖升至9.79mmol／L，较对照组的7.28mmol／L升幅明显，有显著的统计学意义，p=0.000；2)血乳酸明显升高至7.26mmol／L，对照组仅为4.50mmol／L，p=0.013，有统计学意义；3)尽管各氨基酸表现不一，但总体氨基酸水平较对照组的下降明显；4)动脉血气见PCO₂、HCO₃分别降至15.16mmHg、9.11mmHg，同时BE值降至-12.73mmol／L，均较对照组有显著差异，p＜0.01，同期pH值及PO₂不变；5)能量代谢仪监测指标显示高代谢脓毒症时二氧化碳生成量（VCO₂）、氧耗量（VO₂）及能量消耗（EE）分别降至5.46ml／min、6.29 ml／min以及42.88kcal／24hr，与对照组相比p＜0.05，有统计学意义；呼吸商（RQ）轻度升高，但p=0.066，无统计学意义。结论：高代谢脓毒症时机体处于代谢性酸中毒代偿期状态，此时可表现为高。糖、高乳酸血症，血浆总氨基酸水平下降，有氧代谢减弱。第三部分稳定核素示踪法应用于内毒素诱导兔高代谢脓毒血症的代谢研究研究目的：通过第二部分的研究，明确了高代谢脓毒症时机体所处的高糖、高乳酸以及低氨基酸血症；由于机体处于脓毒症早期且代偿能力较强，因此表现为代谢性酸中毒代偿期；通过能量代谢仪的动态检测，我们发现此时机体尽管处于高代谢状态，但主要表现为分解代谢增强，而有氧代谢明显减弱。通过目前的常规方法不能反映此时机体营养底物的流动及代谢情况，文献报道可通过稳定核素示踪法观察活体内各物质的代谢情况，据此我们拟采用¹³C标记的亮氨酸、葡萄糖和乳酸持续灌注法，评价高代谢脓毒症时的氨基酸、葡萄糖和乳酸的代谢情况，并对稳定核素示踪法的应用进行评估。材料和方法：1)分亮氨酸、葡萄糖和乳酸三组各取健康新西兰兔16只，每组再分为高代谢脓毒症组和对照组两部分，建模方法同第一部分；2)三组分别先静脉弹丸式推注再持续灌注¹³C标记的亮氨酸、葡萄糖和乳酸；3)灌注前抽取动脉血作为背景值，灌注达150，160，170和180min分别抽取动脉血2ml用于质谱分析；4)实验开始后前30min中每5min收集呼出气一次，随后每30min收集呼出气一次，用于气相质谱分析；5)血样经过低温离心取上清液，过阳离子及阴离子交换树脂，再经衍生化处理；6)处理过的血样进入气相色谱—质谱仪（GC／MS），测量其¹³C标记的亮氨酸、葡萄糖和乳酸丰度[Ep（％）]；7)实验兔的呼出气通过¹³CO₂气相质谱分析仪测定其中的¹³CO₂丰度(E¹³CO₂)；8)利用文献提供的公式算出CO₂总生成率(V¹³CO₂)以及机体各物质的通量（Q）、氧化速率（C）以及物质代谢百分比（％）等；9)数据分析处理采用t检验。结果：建模且灌注¹³C标记的物质后，结果如下：1)灌注¹³C-Leucine后，高代谢脓毒症组的E¹³CO₂和V¹³CO₂分别为13.53‰和76.79μmol／kg·h，均较对照组显著升高（p＜0.01）；灌注¹³C-Glucose后，高代谢脓毒症组的E¹³CO₂和V¹³CO₂分别为99.16‰和670.29μmol／kg·h，较对照组明显升高，p值均小于0.01，有显著的统计学意义；灌注¹³C-Lactate后，高代谢脓毒症组的E¹³CO₂和V¹³CO₂分别为469.92‰和3050.48μmol／kg·h，均较对照组显著升高（p＜0.01）；2)GC／MS测定结果表明灌注¹³C-Leucine后，高代谢脓毒症组的E_Leucine值为1.02，较对照组明显下降（p=0.000），E_Glucose和E_Lactate分别为0.22和2.87，较对照组显著升高（p值分别等于0.002和0.001）；灌注¹³C-Glucose后，高代谢脓毒症组的E_Glucose值为1.00，较对照组显著降低（p=0.001），E_Lactate值为1.29，较对照组显著升高（p=0.001）；而灌注¹³C-Lactate后，高代谢脓毒症组的E_Lactate值为1.11较对照组明显下降（p=0.002），而E_Glucose则升高至0.36，p=0.001，有显著的统计学差别；3)高代谢脓毒症的的亮氨酸及葡萄糖的通量（Q）分别是408.12和1636.06μmol／kg·h，较对照组明显升高，p值分别等于0.000和0.002，而乳酸的通量（Q）为8632.03μmol／kg·h，虽然绝对值较大，但与对照组相比无统计学意义（p=0.068）；4)高代谢脓毒症组中葡萄糖、亮氨酸以及乳酸的氧化速率（C）值分别为826.75μmol／kg·h、91.7μmol／kg·h和39.03μmol／kg·h，较对照组都出现了可观的升幅，p值均等于0.000，提示与对照组相较有显著的统计学意义；5)高代谢脓毒症的亮氨酸、葡萄糖和乳酸物质代谢百分比（％）分别是22.92％、51.33％及39.03％，较对照组明显升高，p值均小于0.01，有显著的统计学差异。结论：高代谢脓毒症时，亮氨酸、葡萄糖和乳酸的通量（Q）、氧化速率（C）以及物质代谢百分比（％）均较对照组显著升高，证实机体处于高代谢状态；此时机体的分解代谢增强，亮氨酸、葡萄糖转化成乳酸的速率增快，血乳酸升高，同时肝脏糖异生加强，血糖升高；另外，乳酸分解代谢生成CO₂和异生成糖的速率显著加快，且其Q（通量）和C（氧化速率）要远高于葡萄糖和亮氨酸，提示乳酸在此时机体物质、能量代谢中起着不可或缺的作用，其机理有待于进一步探讨。稳定核素示踪法有助于我们了解机体中各种营养物质的代谢改变，有较强的临床参考意义。更多还原

【Abstract】 stage of infection, human body experiences hyperdynamic state with elevated temperature and heart rate, normal or slightly decreased blood pressure, hyper-metabolism or catabolism. So, it is also called hypermetabolic sepsis. The mean body mass decreases rapidly even enough calorie and amino acid is administrated and subsequently impares immune system and prolongs the recovery period. Recent study showed that the pertinence treatment is important. Removing the disease causes, inflammation mediators and hazard metabolic substances together with suitable fluid resuscitation support treatment can prevent the progressing of septic state and can improve the effectiveness of treatment.During hypermetabolic sepsis, the body presents hypermetabolism and catabolism. The decrease of glucose oxidation together with the enhanced glucose anabolism and glucogenesis elevates blood glucose and lactic acid concentrations causing the formation of metabolic acidosis. Enhanced peripheral structural protein catabolism causes increased entrance of amino acids into blood flow induces the increase of amino acids uptaking, transportation and glucogenesis in liver. Clinically it presents negative nitrogen balance, mean body mass loss and impaired immune system. The metabolism of lactic acid, the most important substance causing metabolic acidosis, changes as well. The anabolism increases blood lactic acid concentration for energy providing, glucogenesis. It is important to investigate the interorganic substrates flow for establishing better nutrition support stategy.There are lots of traditional methods to study substances metabolism, such as body weight test, regular blood examination, plasma protein transferron test, resting nutrition assessment, energy metabolism assessment, and dynamic nitrogen balance assessment as well. But all these methods can not tell the detailed flow and path of all kinds of nutrients during altered metabolism. Stable isotope tracing （SIT） can examine the substance metabolism state in life body with higher accuracy, safety and efficacy. It is widely employed in animal and human experiments abroad without any domestic report.This study try to establish an experimental rabbit hypermetabolic sepsis model through the administration of endotoxin. Then inject ¹³C labeled glucose, amino acid, and lactic acid as tracer. After that, examine the concentration of ¹³C labeled CO₂ in exhalation and ¹³C labeled various substance in blood flow so that we can have an idea of the alteration for various substrates flow during hypermetabolic sepsis.The study is divided into three parts:Part 1: Establish rabbit hypermetabolic sepsis model using LPSStudy aim: Sepsis is a dangerous clinical situation. The mortality is very high once it processing to shock stage. So the research emphasis lies on how to block its way to shock. During compensative hyperdynamic stage, the body present impaired nutrients utility ability, confused acid-base balance, and diminished immunity all because of hypermetabolism. It is very important to deep investigate the metabolic flow of all kinds of nutrients for the treatment of such situation. There are all kinds of hypermetabolic sepsis animal models such as rabbit, rodent, sheep and pig as well in the reference reports. We here use rabbit as animal model because they react similarly to endotoxin as homo sabien. Besides, we can obtain enough blood from a rabbit. We used micro-injection pumps continuously inject LPS （endotoxin） so as to form rabbit hypermetabolic sepsis model. The success criteria is that we can find elevated body temperatures , accelerated heart beat, and normal or slightly decreased blood pressure in rabbits.Materials and Methods: 1) Healthy New Zealand rabbits were divided into 2 groups with 8 rabbits each. Rabbits were fastened for 12 hrs with the permission of drinking water as will. 2) Animals were anaesthetized through intermuscular injection of chloramine and diazepam. Then trachea intubation, jugular artery, jugular vein and femoral artery were catherizations were performed. 3) LPS were continuously administrated trough jugular vein at a velocity of 10ng/kg.min for 3hrs. 4) Rabbits body temperature, HR, breath, and BP were read out from multifunctional intensive care monitors. 5) Blood were obtained for blood RT and plasma endotoxin concentration check. 6) All data underwent ANOVA analysis.Results: After 3hrs of LPS injection, 1) in hypermetabolic sepsis （HS） group, the average HR went to 334/min, the average body temperature went to 41.0℃, while in control group they were 274.9/min and 38.2℃ respectively. p=0.000, presented significant statistical differences. The average BP and breath rate in HS group were 97.1mmHg and 94.3/min , while they were 95.9mmHg and 76.4/min in control group, respectively presenting no statistical differences. 2) In HS group plasma endotoxin concentration was 0.088EU/ml while it was 0.013 EU/ml in control group presenting statistical significance, p=0.002. 3) Blood white cell ratio and total white cell counting were 82.73% and 1.90 ×10⁹ in HS group, while they were 64.39% and 7.58×10⁹ in control group presenting very significant statistical differences（p=0.001）. There was no statistical significances in RBC counting and hemocrit between two groups.Conclusions: The rabbit hypermetabolic sepsis model was successfully established through LPS infusing with typical clinical manifestation of accelerated HR, elevated body temperature and unchanged BP.Part II: Metabolic changes happened in endotoxin induced rabbit hypermetabolic sepsisStudy aim: After a period of extreme catabolism during hypermetabolic sepsis, the body develop a serial of hard-to-control clinical situations such as decreased body weight, lowed plasma albumin concentration, anemia, impaired immunity and metabolism disturbance. The metabolism disturbance is a key step which can trigger a cascade of subsequent reactions making the situations much worse. So it is very important to explored the metabolism disturbance situation for improving therapy efficacy. Based on previous depict animal model it is possible for us to better investigate the glucose/ energy, amino acid metabolism and acid-base balance change during hypermetabolic sepsis.Materials and Methods: 1) Animal model and group were depict before; 2) Blood was drew before and 30, 60,120 and 180 min after the initiation of the experiment for baseline and time line blood glucose concentration test. 3) Blood lactic acid concentration were obtain through lactic acid examine kits; 4)Blood amino acid concentration was examined and evaluated through amino acid analyzer. 5) Rabbit baseline and 30, 60, 120, and 180 min post-experimental blood gas value were obtain through automatic blood gas analyzer to evaluate acid-base balance during hypermetabolic sepsis; 6)Rabbits carbon dioxide production （VCO₂）, oxygen consumption （VO₂）, energy consumption （EE）, and breath quote （RQ） were monitored through energy-metabolism analyzer; 7) All data was analyzed through ANOVA.Results: The metabolism change was as follow: 1) The average BG was 9.79mmol/L in HS group compare to 7.28mmol/L in control group presenting statistical significances. 2) Blood lactic acid was 7.26mmol/L in HS group compare to 4.50mmol/L in control group presenting statistical significances; 3) Although every distinct amino acid had their own changes, the total amino acid level was decreased in HS group compare to control group; 4) Artery blood PCO₂, HCO₃, and BE decreased to 15.16mmHg, 9.11mmHg, and -12.73mmol, respectively presenting statistical significances compare to control group, while pH value and PO₂ had no change; 5) The VCO₂, VO₂ and EE were decreased to 5.46ml/min, 6.29ml/min and 42.88kcal/24hr respectively in HS group present statistical significances compare to control group. RQ elevated slightly in HS group with no statistical significances.Conclusions: During hypermetabolic sepsis, the body presents compensated metabolic acidosis state with elevated blood glucose concentration, hyperlactemia, decreased total amino acid level and diminished oxygen metabolism.Part III: Metabolism research in LPS induced rabbit HS through stable isotope tracing techniquesStudy aim: Part II results provided evidences of hyperglycemia, hyperlactemia, and hypoprotemia during HS; at this early stage of sepsis, the body presented strong compensation ability. The internal environment exhibited compensated metabolic acidosis. Although the body experiences hyper-metabolic state, catabolism is the main melody. Oxygen needed metabolism was minimized. So, it is impossible to show real nutrients substrates flow and their metabolism during this stage through regular methods. There were some references reports about using stable isotopes as tracer to find out the substrates metabolic pathway during this stage. So we designed continuous administration techniques using ¹³C labeled leucine, glucose, and lactic acid as tracers to evaluate metabolism situation of different substrates. We also evaluated the efficacy of this method.Materals and Methods: 1) Healthy New Zealand rabbits were divided into three groups with 16 rabbits in each group. In every group, rabbits were subdivided into experimental and control subgroups. 2) Rabbits were bullets injected followed by continuous injected with ¹³C labeled leucine, glucose, and lactic acid; 3) Blood were drewed before and 150,160,170, and 180 min after the initiation of isotopes injection for material analysis; 4) Exhale gas were collected every 5 min in the first 30 min followed by every 30 min there after for material analysis; 5) After centrifuge in low temp the supernatant of the blood samples were collected and went through axon and anon exchange column treatment; 6) Treated blood sample was used for ¹³C labeled leucine, glucose lactic acid examination through mass spectrograph; 7) The exhale gas was collected for ¹³CO₂ exam through gas-phase mass spectrograph; 8) CO₂ total production rate (V¹³CO₂), body various substances production （Q）, oxidation speed （C）, and substances metabolic percentage （%） all can be calculated through equations provided by references below; 9) Data was processed through student t test.Results: 1) After given ¹³C-leucine the E¹³CO₂ and V¹³CO₂ were 13.53% and 76.79umol/kg.h, in HS group respectively elevated significantly （p<0.01） compare to control group. After given ¹³C-glucose, the E¹³CO₂ and V¹³CO₂ were 99.16% and 670.29umol/kg.h, in HS group respectively elevated significantly （p<0.01） compare to control group.After given ¹³C-lactic acid, the E¹³CO₂ and V¹³CO₂ were 469.92% and 3050.48umol/kg.h, in HS group respectively elevated significantly （p<0.001） compare to control group; 2) GC/MS examine showed that after given ¹³C-leucine, the E_leucine value was 1.02 in HS group decreased siginificantly （p=0.000）compare to control group, while E_glucose and E_lactate were 0.22 and 2.87 in HS group elevated significantly （p=0.002 and 0.001） compare to control group; After given ¹³C-glucose, the E_glucose value was 1.0 in HS group decreased siginificantly （p=0.001）compare to control group, while E_lactate was 1.29in HS group elevated significantly （p=0.001） compare tocontrol group; After given ¹³C-lactate, the E_lactate value was 1.11 in HS group decreased siginificantly （p=0.002）compare to control group, while E_glucose was 0.36 in HS group elevated significantly （p=0.001） compare to control group; 3)In HS state, the production （Q） of leucine and glucose were 408.12 and 1636.06 umol/kg.h elevated significantly （p<0.001, p<0.05） compare to control groups. The production （Q） of lactate although had a high value of 8632.03umol/kg.h, presented no statistical significance compare to control groups; 4)The oxidation speed （C） of glucose, leucine and lactate were 826.75, 91.7, and 39.03umol/kg.h respectively in HS group presenting signifigant elevation （p=0.000）; 5) The substances metabolic percentage of leucine, glucose, and lactate were 22.92%, 51.33%, and 39.03% respectively in HS group elevated significantly （p<0.01） compare to control group.Conclusions: During HS, the body experiences hyper-metabolism state proved by elevated leucine, glucose and lactate production （Q）, oxidation speed （C）, and substances metabolism percentage （%）; At this time, enhanced catabolism turns leucine and glucose rapidly into lactate elevating blood lactate concentration. The accelerated glucogenesis in liver elevates blood glucose concentration. In another hand, the accelerated catabolism of lactate into CO₂ and glucose with higher Q and C than that of glucose and leucine suggesting the indispensable role of lactate in substances and energy metabolism. The underline mechanism is waiting for future investigation. The stable isotope tracing techniques is an efficacious method to investigate nutrients metabolic changes in different situations.更多还原