Ion Gradients And Metabolic Energy In Animal Tissue

工作灌注心脏中九种主要无机离子的梯度能量彼此接近平衡，胞外和胞内相之间的电势和ATP水解的DG接近平衡（增田T等，J Biol Chem 1990;265：20321-34）。乙醇的代谢将肝细胞的静息电位从28 mV增加到40 mV（Veech RL等人，Alcoholism Clin Exp Res 1994;18：1040-56）。先前，我们表明，仅仅改变可用的底物就改变了心脏中ATP水解的DG（Kashiwaya Y等人，Am J Cardiol 1997;80：50 A-64 A）。由于任何类型的损伤都会诱导细胞离子分布的定型变化，其中细胞获得Na+，失去K+并肿胀，因此这些损伤的定型变化可能通过简单地改变给予损伤或烧伤受害者的液体组成而逆转。由于这些研究和我们向医学院召集的专家组提出的建议，已经提出了一项建议，即开始研究制造新的复苏液体的可行性（见：液体复苏，治疗战斗伤亡和平民受伤的科学现状，国家学院出版社，1999年）。目标是改善出血和烧伤的标准治疗，这在过去50年中没有改变。我们正在与海军血液研究实验室合作进行这项工作。我们的手稿将ATP水解的Δ G与心脏、肝脏和红细胞的细胞外和细胞内阶段之间的所有9种常见无机离子的梯度能量相关联，现已发表（Veech，R.L.等人，IUBMB Life，54：241-252，2002）。这些组织的电位从-86到-28到-6 mV不等。我们发现，Na+梯度的能量约为ATP水解能量的1/3，并且由KCl微电极测量的静息膜电位是电泳最渗透离子所需的功函数。因此，离子梯度系统似乎是一个吉布斯唐南近平衡系统依赖于ATP水解的能量。这导致制备和测试了一种新的用于出血、复苏、头部创伤、中风和在军事和民用环境中治疗烧伤的肠胃外流体家族，并且已经发表了发现（Liberthal，W.等人，Shock，17：61-69，2002）。在军事环境中，一半的致命伤是头部创伤的结果。因此，我们设计了用于治疗与出血相关的头部创伤的新液体。与海军研究办公室合作，这些新液体目前正在全国四个不同实验室的动物模型中进行评估，以确定这些新液体是否降低发病率和死亡率。我们与牛津大学的BHF NMR实验室合作，通过测定镰状细胞贫血患者红细胞中的游离[Mg 2 +]，继续并扩展了我们在游离[Mg 2 +]测定方面的工作。镁的管理已被提议作为镰状危象的治疗。我们在这篇论文中表明，以前的报告游离[Mg 2 +]水平升高是不正确的，由于ATP的损失，导致Mg 2+结合减少。这些观察结果应该是有价值的，在确定适当的治疗镰状危象。该手稿现已出版（Wilcox，J. P.等人，J.Biol.Chem.277：49911-20，2002）。 用于常规手术、出血和烧伤复苏的复苏液50年来一直没有改变。标准的高容量液体是林格氏液！| s乳酸盐，其含有26 mM D，L-乳酸盐，尽管最近出现了一些仅含有L-乳酸盐的乳酸盐。已经反复证明，d-乳酸的代谢产生不良后果，包括脑病、心律失常（26）和出血后肺中的细胞凋亡（27）。所谓的岘港肺是越南战争中出血治疗期间发病率和死亡率的主要原因。因此，已经制备了一个新的系列，其中D，L-乳酸钠被D-[O-羟基丁酸钠]取代，并在许多出血的动物模型中进行测试，并检查在肺、肾和心脏中的作用。最近，在波士顿进行的大鼠出血模型研究表明，与标准林格氏乳酸盐、0.9%氯化钠或葡萄糖、K+和胰岛素相比，给予林格氏酮溶液可降低大鼠死亡率。这项工作得到了国防部提供的资金的支持，最近，应DARPA的请求，与海军血液研究实验室的Robert Valeri博士合作提交了一份资金申请。如果成功，该计划将进展到这些新液体的临床试验。预计将于2004年10月作出决定。

The energy of the gradients of the nine major inorganic ions in working perfused heart are in near equilibrium with each other, the electrical potential between extra- and intracellular phase and the DG of ATP hydrolysis (Masuda T et al, J Biol Chem 1990;265:20321-34). The metabolism of ethanol increases the resting electrical potential of hepatocytes from 28 to 40 mV (Veech RL et al, Alcoholism Clin Exp Res 1994;18:1040-56). Previously, we showed that merely changing the substrate available altered the DG of ATP hydrolysis in heart (Kashiwaya Y et al, Am J Cardiol 1997;80:50A- 64A). Since injuries of any sort induce a stereotypic change in cellular ionic distributions wherein the cell gains Na+, loses K+ and swells, these stereotypic changes of injury can possibly be reversed by simple changes in the compositions of fluids administered to victims of injury or burns. As a result of these studies and our suggestions to a panel convened by the Academy of Medicine, a recommendation has been made that investigation of the feasibility of making new resuscitation fluids be initiated (see: Fluid Resuscitation, state of the science for treating combat casualties and civilian injuries, National Academy Press, 1999). The goal is to improve the standard treatment of hemorrhage and burns, which has not changed over the past 50 years. We are collaborating in this effort with the Naval Blood Research Lab. Our manuscript relating the Delta G of ATP hydrolysis to the energy of the gradients of all 9 common inorganic ions between extra and intracellular phases of heart, liver and red blood cell has now been published (Veech, R.L. et al, IUBMB Life, 54: 241-252, 2002). These tissues differ in electrical potential from -86 to -28 to -6 mV. We found that the energy of the Na+ gradient was about 1/3 of the energy of ATP hydrolysis and that the resting membrane potential as measured by KCl microelectrodes was a work function required to electrophoresis the most permeant ion. The system of ion gradients therefore appears to be a Gibbs Donnan near-equilibrium system dependent upon the energy of ATP hydrolysis. This has led to the preparation and testing of a new family of parenteral fluids for use in hemorrhage, resuscitation, head trauma, stroke and the treatment of burns in both military and civilian settings and findings have been published (Liberthal, W. et al, Shock, 17: 61-69, 2002). In military settings, half of all fatal wounds are the result of head trauma. Accordingly, we have designed new fluids for the treatment of head trauma associated with hemmorhage. In collaboration with the Office of Naval Research, these new fluids are currently being evaluated in animal models in four different laboratories across the country to determine if these new fluid decrease morbidity and mortality. We have continued and extended our work on the determination of free [Mg2+], in collaboration with the BHF NMR laboratory in Oxford by determining the free [Mg2+] in red cells from patients with sickle cell anemia. Administration of Mg had been proposed as a therapy for sickle crisis. We showed in this paper, that previous reports of the levels of free [Mg2+] being elevated were incorrect due to loss of ATP with result decrease in Mg2+ binding. These observations should be of value in determining appropriate therapy in sickle crisis. This manuscript has now been published (Wilcox, J.P. et al, J. Biol. Chem. 277: 49911-20, 2002). Resuscitation fluids used for routine surgery, resuscitation from hemorrhage and burns have been unaltered for 50 years. The standard high volume fluid is Ringer!|s lactate, which contains 26 mM D,L-lactate, although recently some has appeared containing only L-lactate. It has been repeatedly shown that the metabolism of d-lactate produces untoward consequences including encephalopy, cardiac arrythmias (26) and apoptosis in the lung after hemorrhage (27). So called Da Nang lung was a major cause of morbidity and mortality during treatment of hemorrhage in the Viet Nam war. Accordingly, a new family where Na D,L-lactate is being replaced with Na D-fO-hydroxybutyrate has been prepared and is being tested in a number of animal models of hemorrhage and examining effects in lung, kidney and heart. More recently, studies done in Boston in a hemorrhage model in rat has shown that adimistration of Ringer's ketone solution decreases mortality in the rat when compared to either standard Ringer's lactate, 0.9% sodium chloride or glucose, K+ and insulin. This work has been supported by funds provided by the DoD and more recently, in response to a solicitation from DARPA, a request for funds from has been submitted in collaboration with Dr. Robert Valeri of the Naval Blood Research Laboratory. If successful, this program will progress to clinical trials of these new fluids. A decision is expected in October, 2004.