Ion Gradients And Metabolic Energy In Animal Tissue

在工作灌流心脏中，九种主要无机离子的能量梯度彼此接近平衡，细胞外和细胞内相之间的电势以及三磷酸腺苷的水解度(Masuda T et al，J Biol Chem 1990；265：20321-34)。乙醇的代谢使肝细胞的静息电势从28 mV增加到40 mV(Veech RL等人，酒精中毒临床试验1994；18：1040-56)。以前，我们表明仅仅改变可用底物会改变心脏中ATP水解的DG(Kashiwaya Y等人，Am J心脏ol 1997；80：50A-64A)。由于任何类型的损伤都会引起细胞离子分布的典型变化，即细胞获得Na+、失去K+和肿胀，这些典型的损伤变化可能会被损伤或烧伤患者所用液体成分的简单改变所逆转。由于这些研究和我们向医学学会召集的一个小组提出的建议，建议开始调查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状况，国家科学院出版社，1999年)。目标是提高出血和烧伤的标准治疗，这在过去50年里没有改变。我们正在与海军血液研究实验室合作进行这项工作。我们的手稿现在已经发表了(Veech，R.L.等人，IUBMB Life，54：241-252,2002)，将ATP水解酶的G增量与心、肝和红细胞的胞外和胞内相之间所有9种常见无机离子的能量梯度联系起来。这些组织的电势从-86到-28到-6 mV不等。我们发现Na+梯度的能量约为ATP水解能的1/3，用KCl微电极测得的静止膜电位是电泳膜中最重要的离子所需的功函数。因此，离子梯度体系似乎是一个依赖于ATP水解能量的Gibbs Donnan近平衡体系。这导致了一系列新的肠外液体的制备和测试，这些液体用于军事和民用环境中的出血、复苏、头部创伤、中风和烧伤的治疗，并已发表了研究结果(Liberthal，W.等，Shock，17：61-69,2002)。在军事环境中，所有致命创伤中有一半是头部创伤造成的。因此，我们设计了新的液体来治疗与出血相关的头部创伤。与海军研究办公室合作，目前正在全国四个不同实验室的动物模型中对这些新液体进行评估，以确定这些新液体是否可以降低发病率和死亡率。我们与牛津BHF核磁共振实验室合作，通过测定镰状细胞性贫血患者红细胞中的游离[Mg2+]，继续并扩展了我们在测定游离[Mg2+]方面的工作。服用镁曾被认为是治疗镰刀危机的一种方法。我们在本文中指出，以前关于游离[Mg~(2+)]水平升高的报道是不正确的，这是由于ATP的丢失导致Mg~(2+)结合减少。这些观察结果对确定镰刀危象的适当治疗方法有价值。这份手稿现已出版(Wilcox，J.P.等人，J.Biol。化学。2002年：49911-20)。 用于常规手术、出血和烧伤复苏的复苏液体50年来一直没有改变。标准的大流量液体是林格！|S乳酸，它含有26 mM的D，L-乳酸，尽管最近有一些只含有L-乳酸。研究一再表明，d-乳酸的代谢会产生不良后果，包括脑部病变、心性心律失常(26)和出血后肺组织细胞凋亡(27)。在越南战争期间，所谓的岘港肺是出血治疗期间发病率和死亡率的主要原因。因此，一个新的家族，其中的Na D，L-乳酸盐将被替换为Na D-fo-羟基丁酸酯，并正在许多动物模型中进行出血测试，并检查对肺、肾和心脏的影响。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，放射治疗林格氏酮溶液降低了大鼠的死亡率。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，该计划将进入这些新液体的临床试验。预计将在2004年10月做出决定。

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.