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Madame Curie Bioscience Database [Internet]. Austin (TX): Landes Bioscience; 2000-2013.

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Metabolic Management

*.

* Sufan Chien—Department of Surgery, University of Louisville, Louisville, KY 40202, USA. Email: ten.epacsten@cnafus

The normal human body and the bodies of other mammalian animals have complex neurologic and hormonal regulatory systems. Adequate substrates and oxygen supplies allow these systems to adjust the body's function and its metabolism to a wide variety of external and internal environmental changes. Every organ has a vital role in the control of one or more fluid constituents and the efficient exchange of energy and substrates between organs is crucial for inducing this steady state. Once an organ is separated from the body, the regulatory ability becomes very limited or lost and the survival of the organ becomes dependent upon the correct and careful regulation of the new artificial environment.

Metabolic regulation is involved in nearly all aspects of organ preservation, a subject that is overlapped in other chapters of this book. The scope of this particular chapter varies according to the methods used. If a single-flush preservation is used, the major pathophysiology is reduced energy supply and accumulation of metabolic wastes. Thus, either inhibition of tissue metabolism or supplement of energy substrates along with removal of metabolic wastes become the main strategy for extended organ survival. On the other hand, if perfusion is used, whether it is continuous or intermittent, the above problems become less important and the management concerns shift to overcoming the damage caused by perfusion itself. Our understanding of the physiology of isolated organs is superficial and fragmented because our knowledge of normal organ metabolism is still relatively limited. Moreover, due to the rapid advancement of molecular biology, the metabolic pathway is not currently a favorable research topic,1 leaving many questions unanswered. In this chapter, we will briefly review previous work in these areas plus some findings in the author's laboratory describing his research team's effort to extend organ and tissue survival time during ischemia. Because many topics are also covered in other chapters, these topics will be mentioned only briefly. Metabolic management during reperfusion will not be described here because a separate chapter is dedicated to reperfusion damage.

Ischemic Effects on Tissues and Organs

Solid organs, such as the heart, liver, kidney and brain, require large amounts of oxygen and fuel to support their various specialized functions. Eighty percent of the oxygen consumed by the human body is used for energy production, which is produced when large, complex molecules (such as carbohydrates, lipids and proteins) are broken down into smaller, simpler molecules. The resultant decrease in chemical order causes the liberation of free energy. It is estimated that in humans, the rate of adenosine triphosphate (ATP) synthesis varies daily from 80 moles (about 40 kg) at rest to 1800 moles (∼ 910 kg) during strenuous exercise.2 , 3 In general, glucose metabolism supplies the largest portion of cellular energy through its various steps (i.e., conversion of substrates to acetyl-CoA in the cytoplasm, the oxidation of acetyl-CoA in the Krebs citric acid cycle and electron transfer activities coupled to oxidative rephosphorylation of ADP to ATP in the respiratory chain of the mitochondria, which is responsible for 90-95% of the total ATP requirement in eukaryotic cells).2

A great number of events occur when tissues and organs are removed from the body. As blood circulation stops, there is a combined effect of anoxia, lack of substrate and absence of metabolic waste removal. The tissues use stored glycogen and the leftover oxygen for continuous aerobic metabolism. However, this process soon stops due to depleted oxygen supply. In an effort to maintain tissue energy supply, glycolysis takes over, but this is not an efficient way to produce energy and the process also results in intracellular acidosis. While the pathophysiology of ischemia is complex,4 some of these events have been clearly delineated, such as depletion of high-energy phosphate, inhibition of cellular energy-dependent processes, generation of oxygen-free radicals, loss of osmotic balance across the membrane, stimulation of glycolysis, accumulation of metabolic products, intracellular acidosis and release of lysosomal enzymes.5 , 6 Short episodes of ischemia cause mild damage from which a cell can recover, while longer periods of ischemia cause irreversible cell damage, leading to cell death. An organ's tolerance to ischemia depends on the following three factors: (1) the size of the available stored energy pool; (2) the amount of the energy-demand per time unit; and (3) the efficiency of the anaerobically obtained energy.7 At 37°C, the maximum tolerable ischemic time is about 30 to 45 minutes for the heart,8 , 9 30 to 60 minutes for the liver10 , 11 and less than 5 minutes for the brain.12 , 13 Beyond these time limits, irreversible damage results. Even if blood circulation is reestablished, total functional recovery is impossible.12 , 14 Furthermore, loss of function always precedes cell death. Some ischemic myocardial tissue, for example, can survive for 30 to 60 minutes, but cardiac muscle contractility is lost within less than 1 minute. Similarly, although neurons survive for some minutes, consciousness is lost almost immediately after a sudden interruption of blood supply to the brain.15

Decline of High-Energy Phosphates

The biochemical processes of high-energy phosphate decline are complex and some are still not fully understood. However, it is generally agreed that the following events occur as the result of ischemia:

  1. The depletion of oxygen stores occurs within a few seconds in many organs.
    Our bodies require approximately 500 g of molecular oxygen per day.16 The oxygen is continuously transported to the tissue mainly by oxygen-binding protein hemoglobin. Removal of an organ from the body induces complete tissue ischemia, resulting in shutdown of oxygen and nutrients into the cells. Except for the lungs, residual oxygen in the tissue is very limited and its supply lasts only a few seconds when blood circulation stops. After this interval, oxidative metabolism ceases. For example, in the brain, tissue pO2 falls to 0 in less than 30 seconds.12 , 17 The myocardial oxygen reserve is depleted within 8 seconds following aortic cross-clamping, when the pO2 falls below 5 mmHg.18 The blood oxygen content in the liver or kidney at the moment of separation from blood supply is about 1.5 μmol/g fresh weight of tissue. This would provide less than a 1 minute supply of oxygen for the liver and much less for the kidney, which consumes about 5 μmol of O2/min per gram of tissue.19
  2. Stored energy in the form of creatine phosphate (CP) and adenosine triphosphate (ATP) is reduced and depleted.
    Although there is a progressive decrease of ATP levels after ischemia, the early decrease of creatine phosphate is more striking, especially in the myocardium. When the ATP/ADP ratio declines, ATP is regenerated rapidly by the reversible creatine kinase (CK) reaction to replenish the ATP pool. CP is utilized while the ATP concentration is only slightly reduced from its resting level.20 , 21
    The exact times at which the changes of high-energy phosphates occur are difficult to determine because of the extreme lability of CP and ATP. Data accumulated from decades of research in many laboratories, including ours, have shown that the level of myocardial CP is reduced by 50% within 1 minute after inducing ischemia. After 5 minutes, CP is nearly depleted.22 - 24 ATP levels do not decline as rapidly as CP levels; they decrease to about 50% after 5 to 10 minutes into ischemia.25 , 26
    As oxygen levels continue to fall and oxidative phosphorylation ceases, ATP levels are depleted. Although 40 to 910 kg of ATP is synthesized in the adult human body every day, the storage of ATP in tissues is minimal. For example, the human brain can generate 12 kg of ATP per 24 hours, but has only about 5 mmoles of reserve for all species of adenine nucleotides (ATP+ADP+AMP). No large reservoirs of any energy-rich substances such as ATP or CP exist in any tissue.27 Furthermore, the average ATP molecule has a lifespan of only 1 to 5 minutes and most cells will be depleted of ATP very soon after the cessation of oxidative phosphorylation.15 Five minutes of complete ischemia is sufficient to lower brain ATP practically to zero. The breakdown of ATP increases levels of ADP, AMP and Pi, thereby increasing the levels of adenosine, inosine, xanthine and uric acid. The continued nucleotide degradation to the membrane-diffusible nucleosides results in the inability to rephosphorylate adenosine monophosphate (AMP) when circulation is reestablished and this slow repletion is especially troublesome for some organs that need immediate function such as the heart.28
  3. Anaerobic glycolysis increases in an attempt to maintain normal cellular function.
    When the oxygen supply to the tissue is normal, the rate of glycolysis is inhibited by high levels of citrate and ATP formed by oxidative metabolism in the citrate cycle (TCA cycle). When there is anoxia or severe hypoxia, oxidative metabolism ceases, citrate and ATP levels fall and glycolysis is stimulated. This feedback mechanism is the Pasteur effect.29 Glycolysis is fueled by stored glycogen, whose breakdown also is stimulated by a low energy charge. However, this has low efficiency for energy production. One mole of glucose, when completely oxidized, yields 36 to 38 moles of ATP (depending on the shuttle mechanism by which the NADH enters the mitochondria), whereas the same amount of glucose yields only 2 moles of ATP when metabolized to lactate under anaerobic conditions, approximately 18 times less useful energy is produced as compared to aerobic production.30 , 31 Normally, anaerobic glycolysis is suitable only for cells with low energy requirements and as a temporary device to survive episodes of hypoxia or to cope with increased energy demands. In most tissues (except red blood cells, the cornea, etc.), glycolysis is only a preparatory pathway for subsequent oxygen-requiring metabolism. In the presence of oxygen, the end-product of glycolysis is not lactate, but pyruvate, which then enters the mitochondria and is oxidized to acetyl CoA by pyruvate dehydrogenase. Acetyl CoA is oxidized to CO2 by the TCA cycle.
    Despite its low efficiency, ATP produced from glycolysis and oxidative phosphorylation is not of equal importance. First, carbohydrates are the only metabolic substrates that can produce ATP under anaerobic conditions.15 Because glycolysis can provide energy even in the absence of oxygen, inhibition of the Pasteur effect by severe ischemia limits the capacity of the tissue to survive the ischemic insult.29 Second, glycolysis has a special role in maintaining ion homeostasis. For example, in the heart, aerobically produced ATP supplies energy for contraction, whereas glycolytically produced ATP that contributes only 5 to 10% of the overall ATP supply in normal aerobic state is restricted to processes involved in membrane functions such as ionic pumps and the maintenance of the phosphatidic acid cycle.32 , 33 Only a very limited portion of the aerobically produced ATP is available for similar functions.34 , 35
    However, glycolysis cannot maintain tissue survival for a longer period of time. When there is not only extreme hypoxia but poor blood flow (ischemia), products of glycolysis accumulate, the protons and lactate inhibit glycolysis and glucose use falls. In the heart, it has been observed that the initial phase of rapid glycolysis lasts only 1 to 2 minutes, followed by a slower rate which lasts about 90 minutes. The terminal slowing and final cessation of anaerobic glycolysis occurs when the ATP of the tissue decreases to less than 1.0 μmol/g dry weight.36
    The final cessation of glycolysis is most likely due to the combination of inhibition of glycolytic enzymes, depletion of substrates and cofactors and decrease of tissue pH.36 Lactate is produced by glycolysis under anaerobic conditions. The onset of the maximal rate of lactate formation occurs within 1 minute of severance of blood supply to organs.19 Being an acid, lactic acid tends to acidify the cell in which it is formed. An increased acidity dampens glycolytic activity when pyruvic and lactic acid, the end products of glycolysis, accumulate to dangerous levels and glycolysis is eventually inhibited.15
  4. All energy-dependent functions start to cease.
    Many living cells must transport molecules against their concentration gradients. This is an active transport and energy provided by ATP-hydrolysis is used to drive the uphill transport of molecules in the energetically unfavorable direction. The ion pumps responsible for maintaining gradients of ions across the plasma membrane provide important examples of active transport driven directly by ATP hydrolysis. The concentration of Na+ is approximately 10 times higher outside cells than inside cells, whereas the concentration of K+ is higher inside than out. These ion gradients are maintained by the Na+/K+ pump (Na+/K+-ATPase), which uses energy derived from ATP hydrolysis to transport Na+ and K+ against their electrochemical gradients. The Na+/K+ pump is estimated to consume nearly 25% of the ATP utilized by many animal cells and one of the major functions of this pump is to maintain osmotic balance and cell volume.37 The active transport of Ca++ across the plasma membrane is driven by a Ca++ pump that is structurally related to the Na+/K+ pump and is similarly powered by ATP hydrolysis. The Ca++ pump transports Ca++ out of the cell, so that intracellular Ca++ is maintained to approximately 0.1 μM, in comparison to the extracellular concentration of about 1 mM. In some cells, similar ion pumps are responsible for the active transport of H+ out of the cell.37 Together, these pumps function to maintain membrane gradient. Glycolytically derived ATP is preferentially used for maintaining the ion gradients and the loss of K+ from the cell was shown to be more clearly marked during inhibition of glycolysis.33 , 38 - 40 Similar results were also seen in sodium infux41 and enzyme release despite similar ATP levels.42 , 43 With the declined pump function, membrane gradient disappears and cells swell. If the process is not stopped, death follows quickly.
  5. Metabolic waste accumulation and decrease of cellular pH levels.
    The acidity of the blood is tightly regulated at a pH level of 7.35 to 7.40. A decrease of the blood pH below this range is called acidosis. Lactic acidosis is the most common type of metabolic acidosis and an impairment of oxidative metabolism is the most common cause of this event. The cellular energy charge decreases, PFK becomes activated and a large amount of pyruvate is formed at a time when its mitochondrial oxidation is impaired and the excess pyruvate is converted to lactate. Continued glycolysis depends on the availability of NAD+ for the oxidation of glyceraldehyde-3-phosphate. This is achieved by lactate dehydrogenase, which oxidizes NADH to NAD+ as it reduces pyruvate to lactate. However, pyruvate becomes scarce in ischemic tissues because glycolysis is inhibited not only by NADH but also by excess protons and lactate. Reduced NAD+ availability has been observed in various tissues and organs during ischemia.44 , 45 As oxygen availability inside a cell is decreased, the oxidation of NADH by molecular oxygen, through the electron carrier system, decreases, resulting in an inhibition of the Krebs cycle of mitochondria.46 , 47 If glycolysis reaction does not generate enough NAD+, the glycolytic pathway arrests at glyceraldehyde-3-phosphate and no more ATP is generated. These changes cause mitochondria to cease functioning after a few seconds of severe anoxia.
    The importance of metabolic waste can be illustrated from another viewpoint. In animal experiments, cells have survived “pure” anoxia without interruption of the blood flow longer than ischemic anoxia. This fact points to the importance of accumulating metabolic products, such as lactic acid, in ischemic cell injury.15
  6. Rapid depletion of glycogen levels occurs under anaerobic conditions.
    Glycogen is a polymer of approximately 10,000 to 40,000 glucose residues, held together by alpha-1,4 glycosidic bonds. With a molecular weight between 106 and 107 daltons, it is as big as a complete human ribosome (4.2 × 106 Da). Anaerobic glycolysis from glycogen produces three rather than two molecules of ATP for each glucose residue: the ATP-consuming hexokinase reaction is not required because glucose 6-phosphate is generated by glycogen phosphorylase and phosphoglucomutase, which do not consume ATP .15 , 48 However, glycogen is mainly stored in the liver (up to 6%) and muscle (0.7%) and consumed quickly. Very little is consumed in other organs.49
  7. Mitochondrial ATP generation is blocked because of a lack of molecular oxygen, and insufficient supply of NAD+ also causes the TCA cycle to cease function.
    A decreased energy charge and decreased NAD+/NADH ratio are the initial results of oxygen deficiency. The mitochondrial oxidative pathways are arrested for lack of NAD+. The regulated enzymes of glycolysis and glycogen degradation are stimulated by a low energy charge. The accumulation of lactic acid acidifies the cell. Both the plasma membrane and the organellar membranes are damaged by the increased acidity and by osmotic imbalances that result from the failure of ATP-dependent ion pumps.50 Reactive oxygen radicals also cause mitochondrial damage.51 , 52

Intracellular Acidosis

The precise mechanism that causes tissue acidosis is not completely understood and is controversial. The cause is believed to be the result of several metabolic derangements during severe ischemia, especially the accumulation of protons, NADH and lactate. The role of metabolic products in the development of ischemic damage has been appreciated for many years. In 1935, Tennant and Wiggers53 demonstrated that a reduction in coronary flow had a more profound effect on myocardial contractility than did hypoxia. The early decrease in contractile force of ischemic hearts was associated with increased tissue lactate and H+ and little change in tissue ATP.54 , 55 In an animal study, incubation of thin slices of dog myocardium with 50 mM lactate resulted in mitochondrial changes after 10 minutes, a finding similar to those after 1 hour in ischemic myocardium.56 Thus, high-tissue lactate has been implicated as a factor directly or indirectly causing cellular damage during ischemia,57 although new evidence has indicated a more complex function of lactate.58 The formation of ATP by glycolysis rather than by mitochondria has been found to produce more protons that can cause acidosis.59 Another major source of protons is the continued breakdown of ATP formed by anaerobic glycolysis and, to a lesser extent, from ATP-turnover cycles, such as the triglyceride-free fatty acid (FFA) cycle.60 Intramitochondrial acidosis can also result from the production of CO2 during respiration; CO2 produced in the cytosol from bicarbonate can also penetrate the mitochondrial membrane.61 Protons can be produced from glycolytic ATP turnover and net ATP breakdown.59

There is disagreement regarding the relationship between intracellular acidosis and irreversible cell damage. Conventional textbooks of physiology and medicine state that there is close relationship between these two events. This conclusion has been challenged because there has not been strong enough evidence to support it and some evidence has shown that acidosis may be protective during ischemia.62 In organ preservation-transplantation, metabolic acidosis is generally believed to play an important role in replantation toxemia .63

Osmolar Load and Cell Swelling

With only a few exceptions, the cellular plasma membrane is highly permeable to water molecules, urea molecules and chloride ions because they are considerably smaller than the membrane pores. The volume of a living cell is not constant but changes continuously and the rate per second of diffusion of water in each direction through the pores of a cell is about 100 times as great as the volume of the cell itself.64 , 65 The maintenance of cell volume within a physiological limit requires a precise volume-regulatory mechanism. The energy-requiring transport processes present in the plasma membranes of all animal cells play an important role in the regulation of cell volume.66 All of the ions dissolved in the extracellular and intracellular fluids and other substances, such as glucose, amino acids, free fatty acids and electrolytes, can cause osmotic pressure at the cell membrane. This pressure can be very high, reaching approximately 5400 mmHg (a 230 foot water column).65The plasma membrane of the mammalian cell cannot withstand significant hydrostatic pressure gradients. If the osmolarities of extracellular and intracellular fluids are different, water moves across the plasma membrane and volume change occurs (Gibbs-Donnan equilibrium).67

Cell swelling is an important factor in tissue damage and is the primary cause of poor function after transplantation.68 The mechanisms for the modulation of the transport system at the cell membrane are still poorly understood. At least two factors contribute to cell swelling during ischemia:

  1. A substantial proportion of cell energy, provided by hydrolysis of ATP, is used to support cell membrane active transport mechanisms. As stated above, the enzyme system Na+/ K+-ATPase uses energy to expel sodium from the cell and accumulate K+. Each ion tends to diffuse back down its concentration gradient and the outward extrusion of Na+ takes place against both chemical and electrical gradients. Ischemia depresses the activity of the Na+/K+-ATPase in tissues, so that sodium, chloride and water enter the cell and K+ moves out.69 , 70 The degradation of ATP generates more protons and enhances glycolysis. The intracellular osmotic concentration increases, more water streams into the cell and the cell swells.71 , 72 The process creates a vicious cycle, leading to progressive cellular destruction. After the organ is transplanted, this cell swelling prevents normal blood circulation in capillaries and minor blood vessels, resulting in the so-called “no reflow” phenomenon.73 , 74 The ATPase of vascular endothelium is particularly sensitive,75 increasing the no- reflow phenomenon.
  2. During ischemia, the accumulation of metabolic intermediates occurs because of the absence of arterial flow. Lactate, H+, glycolytic intermediates, inorganic phosphate and creatinine are the principal components of this load and, when their levels are significantly increased, are associated with mild cellular edema.76 Mitochondria swell, mitochondrial reactions are severely limited and finally completely inhibited. The influx of calcium causes precipitation and probably alters cellular reactions. Intracellular pH continues to fall, causing a quantitative reduction of negative charges on proteins by hydrogen ions. This changes the structure and function of regulatory and enzyme proteins. As these irreversible changes progress, the cell dies.77
    The resultant damage to various functions may be different from organ to organ. Even within an organ, various tissues have different sensitivities to ischemia and low temperature. For example, in the kidney, ATPase activity in the cortex is well maintained at 10°C, but its activity in the vascular endothelium is completely inhibited at this temperature.78

Generation and Accumulation of Oxygen Free Radicals

Oxygen free radicals are generated during ischemia and play a major role in tissue damage. The topic of oxygen free radicals is covered in chapter by Suzuki et al of this book.

Stimulation of Intracellular Lysosomal Enzymes

As the membranes of the cell become swollen and ineffective because of osmotic stress and increased acidity, leaking of enzymes and other critical elements occurs. The decrease in intracellular pH stimulates the activity of lysosomal membranes to release proteolyses, lipases and other enzymes, which can digest structural and functional components of the cells necessary for organ viability.72 The release of lysosomal enzymes into the cytoplasm can be detected within 30 minutes after ischemia and cellular enzymes leak into the interstitial space and finally appear in the blood.79 When lysosomal enzymes are released in their acidified environment, they attack cellular proteins, glycoproteins, glycolipids, phosphate esters and other substrates. Some of these hydrolytic cleavages, especially those of carboxylic esters and phosphate esters, further acidify the environment.15 The results of the released enzyme initiates the process of self-digestion within the cell, although the consequences of this process on organ viability is still controversial.79 , 80

Damage to Mitochondria

Mitochondria play a central role in the generation of energy in aerobic tissue. With the loss of oxygen and energy, mitochondria swell and the matrix becomes electron-lucent. The space between the cristase, which is continuous with the intramembrane space, becomes enlarged and breaks and the mitochondrial reactions are severely limited, or completely inhibited, causing a rapid loss of mitochondrial activity72 , 81 The formation of mitochondrial transition pores also allows the release of proapoptotic mitochondrial intermembrane proteins, such as cytochrome c and apoptosis-inducing factors, into the cytosol.82 The swelling of mitochondria in the anoxic condition probably has several causes, such as a failure of oxidative phosphorylation, a rise in inorganic phosphate, a rise in free calcium secondary to failure of the calcium pump in the reticulum and the appearance of free fatty acids caused by breakdown of membrane lipoproteins and phospholipids.25 , 79 The rate of activity loss is dependent on the organ and has been suggested as an important factor leading to loss of organ viability72 , 83

Although the biochemical sequence of ischemia, cellular impairment and death has been well established, the exact mechanisms of mitochondrial damage are not totally clear,83 and it is also unknown at which point the cell reaches the irreversible stage. Short episodes of ischemia cause mild damage from which a cell can recover. Modern techniques have no way of establishing when that critical point is reached in a particular cell. The irreversible stage probably varies markedly between cells and especially between tissues.77

Metabolic Inhibition by Hypothermia

The development of hypothermia is based on the principle that reduced temperature decreases metabolism and oxygen consumption, so that organs can survive longer without nutrient supplements.84 Cooling can hinder the rapid deterioration that occurs in an organ at 37°C when deprived of its blood supply. The precise mechanism by which hypothermia protects the viability of organs is very complex and not entirely understood. It is well known that hypothermia changes the reaction rate of all biochemical processes, especially the enzymatic reaction. This temperature dependence of reaction rates can be expressed by the Arrhenius equation:

Rate = Ae-E/RT

where A is a constant known as the pre-exponential factor, T is the absolute temperature, R is the gas constant (8.31 J/mol/K), E is the activation energy (in J/mol) and e is a mathematical number of 2.71828.

An alternative method, the van't Hoff coefficient (Q10), is derived from the Arrhenius equation. It is the decrease in the rate of reaction expressed as a percentage of the initial rate for a 10°C decrease in temperature. For every 10°C drop in temperature, the rate of a chemical reaction is decreased by approximately 50%. The decrease in the metabolic rate during hypothermia varies for the different metabolic processes, depending on the activation energy of the individual reaction, which can give Q10 values ranging from 1.5 to 4. The Q10 values for enzyme-catalyzed reactions usually fall between 1.5 to 2.5, whereas those for non-enzymatic reactions are usually in the range of 2.0 to 4.0.85 , 86 Experimental findings have indicated that the above estimations are suitable for many tissues and organs. Direct measurements of oxygen consumption by dogs showed a 50% reduction of oxygen consumption at 28°C and a 75% reduction at 20°C.87 The mean value of oxygen consumption for the kidney is 6.26 ± 2.49 ml/min/100 g kidney weight at 39°C. At 30°C oxygen consumption is about 43% of the value at 39°C; at 20°C, it is only 16% of the control rate; and at 5°C, it is less than 5% (Fig. 1).85 , 88 However, hypothermia does not produce uniform inhibition of the cellular processes and the inhibition can vary by as much as 400% from one metabolic process to another after a decrease in temperature from 37°C to 0°C.86 Bretschneider89 suggested the following points of orientation for pure ischemia and for myocardial protection with nonbuffered solutions:

Figure 1. Oxygen consumption of rat tissues at different temperatures.

Figure 1

Oxygen consumption of rat tissues at different temperatures. Reprinted with permission from Fuhrman FA, In: Dripps RD, Ed. The Physiology of Induced Hypothermia. Washington, D.C. National Academy of Sciences, 1956: 50-51.

Q10 for 35° to 25°C = 2.2

Q10 for 25° to 15°C = 1.9

Q10 for 15° to 5°C = 1.6

Q10 for 35° to 5°C = 7

Some biochemical processes, especially those localized to cell membranes, show a step change in reaction rates at certain critical temperatures. These changes have been termed phase transitions and are thought to be the result of a change in the consistency of the cell membrane from fluid to gel. In mammalian tissues, phase transitions often occur at about 25°C to 28°C and may cause disturbed cell homeostasis. Biophysical processes, such as osmosis and water diffusion, are affected by temperature to a lesser extent, resulting in a linear change of about 3% per 10°C reduction in temperature.90 , 91 However, osmolar effects become pronounced as the freezing point of water is approached. Ice formation in tissue concentrates solutes in the residual nonfrozen cytosol, causing marked fluid shifts and membrane disruption. For this reason, the freezing point of water is the physical limit to the beneficial effects of hypothermia, unless protective chemical substances are present. Mammalian tissue will not regain function upon thawing from a frozen state, with the exception of some very simple systems, such as red blood cells.92

The use of hypothermia in various medical applications began centuries ago.87 The protection of hypothermia alone was demonstrated long before various preservation solutions were developed. Hypothermia and freezing techniques were used to store arteries for 1 to 35 days for transplantation early in the 20th century.93 - 95 From the 1920s to the 1940s, kidneys were transplanted experimentally after several hours or even within days of simple hypothermia preservation, but without long-term survival.96 - 98 Observations in the early 1950s confirmed that hypothermia significantly reduced heart and renal ischemic damage,99 , 100 and considerable effort was directed toward total protection of organ function with various types of regional hypothermia. In the late 1950s and early 1960s, it was established that reducing the metabolism of the kidney by cooling enabled it to withstand relatively prolonged periods of ischemia and long-term survival of experimental kidney transplants was reported after 4 to 24 hours of simple cold immersion or refrigeration.101 - 103 The simplicity of hypothermic storage stimulated researchers to use this technique as an alternative to perfusion and simple cold storage quickly gained popularity in the 1960s for the preservation of the heart,104 - 106 lungs107 , 108 and other organs.92 , 109

Limitations of Hypothermia and Single Flush Preservation

Single-flush hypothermia is relatively simple and effective for short periods of preservation. However, hypothermia only delays but does not stop organ deterioration.110 , 111 Several limitations make it difficult to use hypothermia for long-term organ preservation. First, although metabolism is reduced, it is not completely suppressed by temperatures above 0°C, even in the presence of chemical metabolic inhibitors. Approximately 5% of the normal activity at 37°C still remains at 0°C.86 Significant active transport and oxygen consumption persist even at 0°C.106 , 112 - 114 Metabolism continues at a temperature of −60°C, so that even when an organ is completely frozen, metabolism still occurs.92 Second, not all reaction rates are affected to the same extent by hypothermia. The effect of cooling is complex and can uncouple reaction pathways, producing harmful consequences.115 , 116 Third, hypothermia itself also has adverse effects on cell physiology.72 , 117 - 119 Hypothermia suppresses the rate of translocation of adenine nucleotides across the mitochondrial inner membrane, thus decreasing ATP synthesis and causing reduced oxidative phosphorylation.72 , 120 The net result is similar to the damage caused by hypoxia. Rapid cooling and profound hypothermia could be deleterious to cell membranes and their function and rewarming the cells to physiologic temperature after exposure to hypothermia also aggravates the cell injury.82 , 121

For single-flush hypothermia preservation, the following additional limitations are well-recognized: 1) substrates for metabolism are not provided after flushing and a collapsed, blocked microcirculation may remain so until the organ is transplanted, which could result in tissue damage;122 and 2) even though some substrates are added to the preservation solution, metabolic wastes can not be disposed of. Accumulation of toxic wastes in tissue will eventually damage cells, causing irreversible organ malfunction after transplantation.123

Despite these limitations, hypothermia is the only universally accepted method for tissue protection through metabolic inhibition. The use of hypothermia is not only limited to organ preservation, but also in other areas such as cardiopulmonary bypass, head injury, aortic surgery and storage of blood and other cells. For solid organs, hypothermic preservation is used most successfully for kidney preservation. For other organs, longer preservation times are still actively sought. Liver preservation times have been improved by using University of Wisconsin solution, but various types of histologic, functional and metabolic damage have been reported at preservation times ranging from 8 to 24 hours. This damage includes loss of adenine nucleotides,124 , 125 damage to sinusoidal endothelial cells126 , 127 and rapid increase in the levels of liver-related enzymes, such as AST and ALT, after transplantation.128 Greater posttransplant problems, including the need for retransplantation, have also been reported for livers preserved for more than 20 hours.

We can speculate that current hypothermic storage times will likely be increased further and that the number of preservable organs will be expanded when more effective preservation solutions are developed. Because of the limitations mentioned above, it is unlikely that single-flush hypothermic storage will provide long-term preservation.

Inhibition of Tissue Metabolism by Hibernation

Metabolic rate depression is an important survival strategy for many animal species and a common element of hibernation, torpor, aestivation, anaerobiosis, diapause and anhydrobiosis.129 - 132 Hibernation in mammals is a unique circannual adaptation allowing certain species, such as the ground squirrel, woodchuck, brown cave bat, European hedgehog and black bear, to survive extended periods of food deprivation when ambient temperatures may be well- below freezing. For example, hibernating ground squirrels conserve up to 90% of the energy that would be required if they remain active during the winter.133 Moreover, it has also been suggested that hibernating animals age at a slower rate than those of the same species that are prevented from undergoing hibernation.134 Profound metabolic changes accompany hibernation, including respiratory depression, a decline in body temperature and a cessation of feeding and renal function. These changes may be of great survival benefit to animals that can subsist without food and water for up to 5 months (up to 8 months in the arctic ground squirrel). In most winter hibernators, except the black bear, body temperatures decline to 4° to 6°C and even 1° or 2°C below freezing in the arctic ground squirrel.135 It is notable that low temperature is not always required to save energy in some animals: hibernation also occurs in summer (aestivation), during periods of excessive drought and heat. Cactus mice, snails, Texas tortoise and some fish and crabs can hibernate in the summer time.136 - 138 These animals can save energy at very high temperatures. However, we know too little about the mechanisms related to aestivation.

Blood or its components from winter hibernating animals have been shown in studies to induce behavioral and physiological depression, including hypothermia, bradycardia, long-term hypophagia without significant weight loss, an anesthetized state and decreased renal function.139 , 140 Kidneys from hibernating animals were kept viable for up to 10 days.141 Studies have also shown that the erythrocytes in hibernating animals have an increased resistance to hemolysis and higher levels of unsaturated fatty acids in the cold blood taken during hibernation.142 Neither human cells nor ground squirrel cells would agglutinate in ground squirrel serum at low temperatures. During hibernation, stored fat becomes the primary metabolic fuel. Carbohydrate needs are met by gluconeogenesis from amino acids. Urea, the primary nitrogen-containing waste product of protein catabolism, is recycled rather than excreted, thereby negating the need for urination and hence arousal.143 , 144 Actively dividing cells, such as those of the intestinal epithelium, become relatively quiescent.145

Despite the profound metabolic, biochemical and cellular changes noted in hibernating animals, relatively little information is available concerning the mechanism(s) that induce and maintain these changes or those involved in reversing them. One major reason for this problem is that most hibernation studies rely on in vivo systems, making experiential manipulation both difficult and expensive. For isolated organ preservation, it may not be necessary to induce whole- body hibernation. In the past, we have attempted to use the hibernation principle for the extension of isolated organs with some success.146 - 149 Subsequent experiments have also indicated protective effects using similar approaches.150 , 151 Plasma from hibernating animals and opioid agonists have both been used to induce a hibernation-like status in nonhibernating animals, but so far no single chemical or chemical combination has been able to induce true hibernation in either whole animals or in an organ. Thus, the theoretical advantages of inducing hibernation in organ preservation have not been successfully translated into the practice. Further characterization of the factors that induce both winter and summer hibernation may prove to be valuable toward extending organ preservation.

Metabolic Substrate Supplement

Supplementation of metabolic substrates to enhance high-energy phosphates represents a more active approach and efforts in this area have been ongoing for decades. The following are several major chemical groups that have shown protective effects in various scenarios. Our research group has focused on the use of fructose-1,6-bisphosphate (FBP, or fructose-1,6-diphosphate, FDP) and direct intracellular delivery of ATP. A more detailed report will be presented here.

Adenosine

During preservation, energy turnover influences the degradation process that affects structural integrity. Adenosine has been shown to enhance the preservation of the heart,152 liver,153 and pan-creas.154 Pharmacologic effects of adenosine are complex because many of the effects are related to adenosine receptors155 , 156 and/or preconditioning,152 rather than simply as a precursor of adenine nucleotides. Other protective effects such as vasodilation, anti-oxidants and inhibition of platelet aggregation have also been proposed.157

Amino Acids

Amino acids may be involved in many aspects of intermediary metabolism, such as protein synthesis, production of substrates for the tricitric acid cycle, provision of buffering properties and promotion of anaerobic ATP production.158 Several amino acids, such as histidine, glutamate, aspartate, arginine and glycine, have been shown to improve heart,159 liver,160 lung,161 intestine162 and kidney163 preservation.

Glucose and Glycogen

Adding glucose or glycogen may enhance organ preservation by maintaining the small rate of metabolism that exists at low temperatures.164 Metabolic substrates are particularly important if a temperature higher than 4°C is the optimum temperature, such as for lung storage. Both glucose and glycogen have been used with some success.162 , 165 Low-potassium dextran solutions enhanced by substrate or glucose-enriched blood were reported to yielded excellent results in some lung preservation studies.166 - 168

NAD and NAD Precursors

Because the decreased NAD+/NADH ratio is the main restraint of anaerobic glycolysis, adding NAD or its precursors may help restore the NAD+ content. In fact, NAD+, nicotinamide, nicotinic acid and quinolinic acid, have been reported to improve myocardial contractility or skin flap survival.169 , 170

Phosphoenolpyruvate (PEP)

Phosphoenolpyruvate is a high-energy metabolite in the final step of glycolysis. PEP is converted into pyruvate by pyruvate kinase and one molecule of ATP is generated. The intact plasma membrane is considered to be impermeable to PEP under normal conditions, but it has been suggested that a change in membrane integrity may occur during ischemia.171 , 172 Furthermore, a small amount of exogenous ATP may transitorily alter the plasma membrane permeability, making it permeable to otherwise nonpermeating substances.173 , 174 The combined use of PEP and ATP during ischemia and in the reperfusion phase was reported to have beneficial effects,175 , 176 along with higher content of pyruvate at the end of ischemia, indicating that PEP was indeed metabolized to pyruvate and to some extent, also to lactate.177 , 178

However, the protective effects have not been duplicated in other studies.177 , 179 It is possible that the altered plasma membrane integrity that enables PEP to enter the cell may also lead to transitory leakage of intracellular constituents such as nucleotides. Our laboratory tested PEP in both cell culture and isolated heart preservation, but we did not find any signs that PEP could penetrate the cell membrane. We did not see a distinctive protective effect in our isolated heart preservation study.

Pyruvate

Pyruvate becomes scarce in ischemic cardiac muscle because glycolysis is inhibited not only by NADH but also by excess protons and lactate. Thus, exogenous pyruvate may help to regenerate NAD+. Infusion of pyruvate was reported to reduce NADH levels and exert a protective effect in heart and liver preservation.180 , 181

AMP Precursors

With the rapid cessation of oxidative phosphorylation during ischemia, not only ATP but also ADP and AMP levels decrease quickly.18 , 182 This decrease is the result of the further breakdown of AMP to adenosine, inosine, hypoxanthine, adenine and purines. Hypoxia in the heart results in a marked increase in the above nucleosides and purines and the amounts of these released products are inversely proportional to the oxygen tension of the perfusion fluid.183 , 184 The major difference between AMP and the nucleosides and purines is that these latter substances can easily be transported through the cell membrane and lost in the preservation solution or perfusate,183 , 185 thereby being unavailable for the nucleotide salvage pathways.186 , 187 The cell is now dependent on very slow and energy-consuming de novo synthesis.188

Adding AMP precursors such as adenine, ribose, hypoxanthine and inosine has been reported to improve organ function.183 , 184 Ribose, a pentose sugar, can be phosphorylated directly to ribose -5-phosphate and then to phosphoribosyl pyrophosphate (PRPP). It can augment both the de novo and salvage pathways.189 , 190 The combined use of ribose and adenine has been shown to shorten ATP resynthesis time dramatically in several reports.190 , 191 Using temporary LAD ligation, Zimmer and Ibel192 showed that continuous intravenous infusion of ribose during recovery from a 15-minute period of myocardial ischemia in rats led to restoration of the cardiac ATP pool within 12 hours, whereas 72 hours were needed for ATP normalization without any intervention. Using a chronic dog model, St. Cyr et al189 , 191 reported that ATP levels continued to fall during the first 4 hours of reperfusion and the eventual recovery from global ischemic insult was prolonged. The mean recovery rate for ATP levels was about 0.04 nmoles/mg wet wt/day and the projected complete recovery time was 9.9 days (9-14 days). Infusion of adenine and ribose enhanced ATP recovery rate to 2.44 nmoles/mg wet wt/day and recovery was virtually complete (96%) by 24 hours. Similar results were also shown by Mauser et al183 and Boldwin et al193 However, mixed results were also reported by others,190 , 192 and limited experience in our laboratory has not confirmed any significant protective effect. This may be because the synthesis of the purine ring, in addition to ribose, is also partly rate-determining for restoration of adenine nucleotide.194

Insulin and Dichloroacetate (DCA)

During tissue ischemia, the final product of glycolysis is not pyruvate, but lactate. Accumulation of a higher level of lactate has detrimental effects on ionic homeostasis. It inhibits glycolysis itself,195 , 196 increases free radical production, induces intracellular acidosis and stimulates calcium release from intracellular stores.197 This can partly explain the supposition that glycolysis is deleterious to the ischemic heart57 , 198 and is another reason why continuous organ perfusion offers better preservation.72 , 199

Insulin and DCA have been used to reduce lactate accumulation because of their ability to stimulate pyruvate dehydrogenase (PDH). The PDH system is a multi-enzyme complex located within the mitochondrion. Tree enzymes in the complex catalyze the stepwise conversion of pyruvate to acetyl-CoA and carbon dioxide, with the first step being the irreversible decarboxylation of pyruvate. PDH is regulated by substrate activation, end product inhibition and reversible phosphorylation. In the presence of Mg++ and ATP, pyruvate dehydrogenase kinase phosphorylates and inactivates the dehydrogenase, while PDH phosphatase reverses this inhibition. Insulin stimulates PDH indirectly by activating the phosphatase, thus increasing the dephosphorylated, catalytically active form of PDH.200 , 201 Insulin treatment decreases extracellular lactate release and improves the preservation of ATP.202 DCA, however, exerts similar effects by inhibiting the kinase.203 , 204 The protective results have been reported in several organs,205 , 206 but negative effects have also been reported.207

Fructose-1,6-Bisphosphate (FBP)

Of all the glycolytic intermediates, fructose- 1,6-bisphosphate (FBP) has received substantial attention for organ preservation and other ischemic conditions. The glycolytic process (Embden-Meyerhof pathway) can be divided into two important phases. In the first phase, glucose is converted to FBP. Two of the enzymes catalyzing the reactions in this phase, hexokinase and phosphofructokinase (PFK), consume 2 ATP molecules. In the second phase, two molecules of glyceraldehyde 3-phosphate are ultimately metabolized to pyruvate and lactate with the production of 4 ATPs per glucose. Thus, the net production of one glucose molecule is 2 ATPs from glycolysis. If FBP were to enter the cell and be used as a substitute for glucose, it enters the glycolytic pathway beyond the two ATP-consuming steps and the two ATP molecules are saved.208 This results in double the ATP production in glycolysis. FBP has been used in various ischemic conditions, including hypothermic preservation of the liver,209 intestine,210 skin flap,211 isolated heart perfusion,212 , 213 neuroprotection,214 circulatory arrest215 and sepsis.216 Increased ATP production has also been reported by many investigators after using FBP.217 , 218

The conventional view portrayed in biochemistry textbooks is that phosphorylated sugars such as FBP cannot cross the cell membrane. Although there have been some studies suggesting that FBP can be metabolized,218 , 219 there is no direct evidence that FBP can actually cross the cell membrane.

Our laboratory has studied the potential protective effect of FBP in cardiomyocytes and isolated heart preservation and beneficial effects were observed.220 - 222 We were especially interested in its possibility of enhancing glycolytic energy production. The following study was carried out to determine, in the absence of glucose, whether FBP could improve myocardial high-energy phosphate metabolism during hypothermic heart preservation. Our results indicated that adding FBP to St. Thomas solution did, in fact, improve high-energy phosphate reserves during hypothermic rabbit heart preservation.

Forty-two adult New Zealand White rabbits were used. Ten were used for normal controls (normal group), in which the hearts were biopsied fresh (without preservation) to obtain normal values of high-energy phosphate. Thirty- two rabbits were divided into two preservation groups for high-energy phosphate study. The animals were anesthetized with an intramuscular injection of ketamine (50 mg/kg), xylazine (5 mg/kg) and chlorpromazine (0.5 mg/kg). The hearts were removed and preserved in cooled St. Thomas solution. In the study group (n = 15), FBP (5 mM) was added to St. Thomas solution. In the control group (n = 17), fructose (5 mM) was added instead. The hearts were preserved at approximately 4°C in a temperature-controlled refrigerator (Model 252, Sanyo Electric Co., Japan) for 18 hours.

After preservation, myocardial samples were taken from the left ventricle (LV), right ventricle (RV), left atrium (LA) and right atrium (RA) separately and high-energy phosphate and its metabolites were measured using HPLC on a Waters 2690 HPLC system with a Waters 996 photodiode array detector and Waters Millenium 32 manager software.

After 18 hours of hypothermic preservation, LV myocardial ATP concentrations declined in both the study and control groups as compared with normal hearts. The LV ATP content in the study group (1.45 ± 0.11 μmol/g wet weight) was about one third of the LV ATP in the normal group (4.42 ± 0.36 μmol/g WW, p < 0.05). However, LV ATP content in the control group (0.65 ± 0.12 μmol/g WW) was only 14% of the LV ATP in the normal group (p < 0.05, Fig. 2). ATP contents in the RV had a similar pattern (p < 0.05 study vs control). ATP contents in the atria also had similar patterns, but the difference between the study and control groups was not as statistically significant as in the ventricles. ADP content was also decreased, but the reduction was not as dramatic as the ATP. LV ADP content was still about 80% of normal in the study group, whereas it was only about 54% of normal in the control group (p < 0.05, study vs control, Fig. 3). ADP content in the RV had a similar change. AMP increased in the hearts after 18-hour preservation. In the control group, AMP levels in the LV increased to 156% of normal. It was also higher than that found in the study group (p < 0.05, Fig. 4). AMP concentration in the RV of the control group was nearly twice as high as that in the normal and study groups (p < 0.05). Total energy (TE = ATP + ADP + AMP) decreased in both preservation groups. In the study group, total energy (TE) in the LV was 58% of that in the normal group, but was still higher than that in the control group, despite the fact that the control group had a very high AMP level (p < 0.05, Fig. 5). The change in energy charge [EC = ( A T P + 1/2ADP)/(ATP + ADP + AMP)] exhibited similar patterns as that of ATP. The energy charge (EC) in the LV (0.58 ± 0.03) and RV (0.65 ± 0.03) in the study group was higher than those in the control group (0.37 ± 0.04 for LV and 0.43 ± 0.03 for RV, p < 0.05, Fig. 6). The ratio of ATP/ADP in the study group was 40% of that in the normal group, but the ATP/ADP ratio in the control group was about 25% of that in the normal group (p < 0.05).

Figure 2. Tissue adenosine triphosphate (ATP) contents after 18-hour preservation in the study (FBP) and control (fructose) groups compared with the normal hearts.

Figure 2

Tissue adenosine triphosphate (ATP) contents after 18-hour preservation in the study (FBP) and control (fructose) groups compared with the normal hearts. In the study group, approximately 33% of normal ATP remained after preservation in each heart chamber, (more...)

Figure 3. Comparison of tissue adenosine diphosphate (ADP) contents in the study (FBP) and control (fructose) groups compared with normal hearts.

Figure 3

Comparison of tissue adenosine diphosphate (ADP) contents in the study (FBP) and control (fructose) groups compared with normal hearts. There was approximately 80% of normal ADP content in the ventricle in the study group and approximately 60% of normal (more...)

Figure 4. Comparison of tissue adenosine monophosphate (AMP) contents in heart chambers among the 3 groups.

Figure 4

Comparison of tissue adenosine monophosphate (AMP) contents in heart chambers among the 3 groups. Note the higher tissue contents of AMP in the ventricle in the control (fructose) group.

Figure 5. Profile of total energy in the study (FBP) and control (fructose) groups compared with the normal hearts.

Figure 5

Profile of total energy in the study (FBP) and control (fructose) groups compared with the normal hearts. About 50% of total energy is present in the study group.

Figure 6. Profile of energy charge among the 3 groups.

Figure 6

Profile of energy charge among the 3 groups. Approximately 80% of energy charge is still present in the study (FBP) group.

When the profile of high-energy phosphates was studied, adenine nucleotides in the four heart chambers in the study group maintained a similar pattern as in the normal hearts. ATP contents increased stepwise from RA, LA, RV, to LV. ATP was higher than ADP, which, in turn, was higher than AMP. However, in the control group, the high-energy phosphate profile was different: the content of ATP was similar in all chambers, but the ratio of ATP:ADP:AMP was reversed (Fig 7).

Figure 7. Comparison of adenine nucleotide profile among the 3 groups.

Figure 7

Comparison of adenine nucleotide profile among the 3 groups. After 18-hour preservation, the profile of adenine nucleotides in each heart chamber in the study (FBP) group is similar to that in normal hearts, but this profile is different or even reversed (more...)

In both preservation groups, the trivial amount of adenosine, xanthine and uric acid was nearly immeasurable in our HPLC analyses. However, IMP, inosine and hypoxanthine levels were increased in all four heart chambers. This increase was especially prominent in the control group.

This study clearly indicated that adding FBP to St. Thomas solution attenuated the decrease of high-energy phosphate reserves during hypothermic rabbit heart preservation.

Our results showed that after 18 hours of preservation, the LV still maintained about 30% of normal ATP levels and about 80% of normal EC in hearts treated with FBP. What do these values tell us? Although no causal relationship has been demonstrated, studies of myocardial ischemia have shown that when there is 80% depletion of myocardial ATP during the ischemic period, irreversible myocardial injury does not occur and adequate reperfusion should result in the eventual return of normal structure and function.55 , 223 When ATP levels are depleted more than 80% (between 20% and 10% of control), disturbances in cellular biochemical function and homeostatic mechanisms occur. Greater than 90% depletion of ATP levels (below 10% of control) is strongly correlated with irreversible myocardial injury.55 , 224 In vivo and in vitro heart studies have also indicated that an EC above 0.60 is generally associated with reversible myocardial injury, but an EC below 0.30 is associated with irreversible injury.55 If these assumptions hold true in rabbit hearts, it may imply that when FBP is added for hypothermic rabbit heart preservation for 18 hours under hypothermia, although stunned, the myocardium may recover after proper reperfusion. This assumption is overly simplified, however. Long-term ischemia studies have shown that ATP levels continue to fall during the first 4 hours of reperfusion after global ischemia and a complete recovery may take as long as 10 days.189

FBP is believed to have multifaceted beneficial effects, including augmentation of high-energy phosphate production during hypoxia,225 , 226 prevention of ischemia/reperfusion-induced leukocyte adherence,227 reduction of lipid peroxidation and oxygen free radical production,228 stimulation of nitric oxide synthase,229 increased red blood cell deformability and decreased blood viscosity,230 chelation of extracellular calcium231 and prevention of platelet activation.232 Many of the proposed beneficial effects of FBP can only be achieved in vivo, in the presence of blood, or during re-oxygenation. Our results were obtained in isolated hearts without the presence of blood or reperfusion. Thus, many of these effects can be excluded from consideration in our rabbit study. Augmentation of glycolytic energy production by FBP has been the most elusive and potentially significant mechanism; it remains the most logical explanation for our results.

Although the true mechanism by which FBP enhances glycolytic energy production is not clear, at least four possibilities have been proposed: (1) direct participation of FBP in glycolysis,218 (2) indirect participation in glycolysis,233 (3) enhancement of glycolysis by activating glycolytic enzymes directly234 and (4) activation of glycolytic enzymes indirectly.235 Except for the first mechanism, the three other possible mechanisms can occur only in a solution containing glucose such as Euro-Collins solution or GIK solution. The St. Thomas solution used in this experiment did not contain glucose and any residual extracellular glucose would have been consumed shortly after the heart was removed. The enhancement of glycolysis induced by FBP would have to occur by the direct participation of FBP in glycolysis and this would require FBP to cross the cell membrane. However, the conventional view is that phosphorylated sugars, such as FBP, do not cross cellular membranes easily. In the past, a few studies have examined FBP movement through the cell membrane, but these studies provided little direct evidence to support this hypothesis. Results from our laboratories have provided some direct evidence that FBP does cross cell membrane.236 , 237 Several possible mechanisms have been proposed recently to explain this special feature of FBP. One mechanism is that FBP affects membrane stability, which allows normally nonpermeant FBP to passively diffuse across the membrane bilayer.236 - 238 This possibility was not explored in the past, but results from our laboratories have indicated that FBP can passively diffuse through artificial membrane bilayers and cellular membranes.236 Although the amount of FBP that can passively move through the membrane bilayers is small, this amount is still higher than normal FBP in the cytosol. Since the concentration of free FBP in the cytosol is only 1.2 μmol/L, an infusion of FBP in the millimolar concentration range results in a more than 1000-fold gradient across the plasma membrane.219 Another mechanism is that FBP might be transported by a dicarboxylate transport system, because its utilization continues to increase at the highest concentration and its transport is inhibited by the dicarboxylate fumarate.239 Our more recent studies have also indicated that in isolated cardiomyocytes at 21°C, FBP uptake exceeded the uptake of L-glucose by several folds. This movement appeared to be via at least two distinct protein-dependent processes.222 Because of its stability, ease of use and low cost, FBP may be a valuable metabolic intermediate for use as an adjacent chemical to improve organ function. More research should clarify some of the possible mechanisms.

Direct Supplement of ATP

Providing high-energy phosphate directly to the ischemic tissue is certainly an attractive idea. In fact, this has been an active project for more than a half- century. Direct infusion of free ATP or other high- energy phosphates would be a simple solution if it could be done. ATP entry into muscle cells was suggested in the 1944 study of Buchthal et al,240 but later work showed that ATP did not enter these cells.241 Because the idea is so attractive, this finding did not discourage others from trying again. Indeed, administration of either free ATP or CP has been reported to improve tissue protection during ischemia in various models and species, such as in the heart,242 kidney,243 liver,244 brain245 and also in various forms of shock.246 , 247 The theory is that since a significant amount of ATP is released into the extracellular space by several types of cells (myocytes, cardiomyocytes, brain cells), ATP can cross cell membranes.248 , 249

However, this theory has not gained wide acceptance in the scientific community; experimental and clinical studies have not duplicated the many promising results. As described below, strongly charged molecules like ATP normally cannot pass the cell membrane without a transporting mechanism.242 , 250 Besides, the half-life of ATP in the blood is less than 40 seconds, which poses technical difficulties for maintaining the ATP supply.251

All cells are surrounded by a plasma membrane that is composed of lipids, proteins and carbohydrates. The basic structure of the plasma membrane is the phospholipid bilayer, which is impermeable to most water-soluble molecules and is responsible for the basic function of membranes as barriers between two aqueous compartments. The plasma membranes of animal cells contain four major phospholipids (phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, sphingomyelin). These phospholipids are asymmetrically distributed between the halves of the membrane bilayer. The internal composition of the cell is maintained because the plasma membrane is selectively permeable to small molecules. Larger, especially charged molecules, for which no specific transport mechanisms exist, cannot cross cell membrane under normal conditions. Specific transport proteins (carrier proteins and channel proteins) are needed for the selective passage of these molecules across the membrane (Fig. 8). Unfortunately, ATP and many energy-rich glycolytic intermediates belong to this category.252 Although the presence of equimolar MgCl2 reduces the negative charge of ATP from 3 to 1, this does not alter the permeability of ATP because no known transporting mechanism exists in the cell membrane. Results from our laboratory have indicated that a small amount of free ATP can be taken up by the cells, most likely through some pores (about 0.8 nm in diameter) in the cell membrane.65 The question is whether this small amount can support tissue metabolism.

Figure 8. Permeability of the cell membrane phospholipid bilayers.

Figure 8

Permeability of the cell membrane phospholipid bilayers. Gases, hydrophobic molecules and small polar uncharged molecules can diffuse through phospholipid bilayers. Larger polar molecules and charged molecules such as ATP cannot. Reprinted with permission (more...)

For the past half century, using various carriers to deliver impermeant substances into the cytoplasm of cells has been a major research interest for cell biologists and clinicians. The ideal system for such delivery would couple both specificity and the ability to easily target multiple cells. Five primary approaches have been used to bypass the plasma membrane barrier: permeabilization, microinjection, polymer delivery, liposomal encapsulation and microemulsion. Permeabilization, which allows entry of materials found in the external medium, has been attained by electroporation and by pore-forming proteins or detergents.253 , 254 However, the lack of specificity and difficulty in controlling this technique limit its application predominantly to research. Furthermore, as soon as nonspecific pores are formed in the cell membranes by permeabilization, many unwanted molecules can also food in, loading the cells with toxic chemicals and losing important membrane ion gradients. While micro-injection allows a more targeted delivery than the permeabilization technique,255 , 256 this is a time-consuming and technically challenging procedure. This procedure, therefore, has limited clinical application. The recent development of biodegradable polymers may offer a new approach for this purpose,257 , 258 but at this time, the main interest is focused on the sustained release of some drugs because polymers are too large to penetrate the cell membrane.259 , 260 Microemulsion can produce much smaller oil droplets, making efficient oil-in-water delivery of drugs.261 , 262 However, ATP is highly hydrophilic and does not dissolve in oil.

One promising approach is the loading of medication into liposomes, or multilamellar vesicles, which are microscopic sacs made of the very phospholipids that constitute cell membranes. When a high enough concentration of phospholipids is mixed with water, the hydrophobic tails spontaneously herd together to exclude water. In contrast, the hydrophilic heads bind to water. The result is a bilayer in which the fatty acid tails point into the membrane's interior and the polar head groups point outward facing the water. As a liposome forms, any water-soluble molecules that have been added to the water are incorporated into the aqueous spaces in the interior of the spheres, whereas any lipid-soluble molecules added to the solvent during vesicle formation are incorporated into the lipid bilayer.263 Liposomes can be filled with a variety of medications and, because of their similarity to cell membranes, are not toxic. They also protect their loads from being diluted or degraded in the blood. As a result, when the liposomes reach diseased tissues, they deliver concentrated doses of medication. Individual liposomes were shown to be capable of carrying tens of thousands of drug molecules, making them an efficient carrier for delivery of certain drugs.264 However, three major problems have limited the widespread use of liposomes for cytosol drug delivery: (1) liposomes are not readily fusogenic, mainly because the stored energy of the vesicles radius of curvature is minimal and the internal layers may inhibit fusion.265 , 266 (2) Because of their size, most intravenously infused liposomes are unable to leave the general circulation, except in areas where vessels become leaky (such as inflammation).264 (3) The body's immune system recognizes the liposomes and removes them from circulation, regardless of the vesicles' composition and size. The liver, spleen and bone marrow take up nearly all liposomes given intravenously, preventing liposomes from circulating long enough to reach many targeted cells and tissues effciently.264 To circumvent these problems, various techniques have been developed such as coating liposomes with polyethylene glycol,267 polyvinyl alcohol,268 or other polymers269 to make them “stealth” and combining liposomes with hemagglutinating virus of Japan (HVJ) to enhance fusion.270

Our laboratory has focused on specially formulated, highly fusogenic and very small unilamellar lipid vesicles (with diameters around 120-160 nm) to encapsulate Mg-ATP. Lipid vesicle fusion (and the delivery of ATP) occurs primarily by one of four methods: (1) increasing electrostatic interactions; (2) destabilizing the membrane bilayer; (3) increasing nonbilayer phases; or (4) creating dissimilar lipid phases. By changing the phospholipid composition through the use of one or all of these methods, a highly fusogenic lipid vesicle can be created: altering the charge of the phospholipid head group, increasing mean molecular area of the lipids, creating dissimilar regions of lipids and increasing the kinetic energy of the lipid vesicles. The fusogenicity of vesicles is strongly influenced by the dynamic and structural properties of the lipid membrane. The existence of a heterogeneous lipid-bilayer structure, which is composed of fluctuating gel and fluid domains that are prevailing in the temperature range of the main phase lipid membrane transition, leads to a strong increase in the fusion rate. Additionally, membrane curvature stress and the incorporation of various nonlamellar forming agents into the lipid membrane has a dramatic effect on the fusion properties.271 Several formulations have been tested and the chemicals used to make ATP-vesicles included L-α-phosphatidylcholine (Soy PC), Mg-ATP, polyethylene glycol, chloroform and trehalose. Our unilamellar vesicles were found to be highly fusogenic. In our study of endothelial culture, it was clearly shown that these vesicles fused with the cells in 5-10 minutes, delivering water-soluble carboxyfuorescein (Fig. 9). In one rat study, primary cardiomyocytes were incubated in either (1) ATP-vesicles, (2) lipid vesicles only, (3) Mg-ATP (5 mM), or (4) culture medium. After equilibration, potassium cyanide (KCN, 5 mM) was added to the culture media for 30 minutes. Results indicated that cardiomyocytes after 30 minutes of chemical hypoxia had the highest ATP and TE contents in the group treated by ATP-vesicles. Neither vesicles alone nor ATP alone had a similar effect (p < 0.05, Fig. 10). The redox status of cardiomyocytes was decreased by the use of KCN. However, in the ATP-vesicles group, this decrease of redox status was significantly different from the other groups. The Alamar-Blue fluorescent intensity reading with ATP-vesicles was higher than with vesicles only, free Mg-ATP only, or with M199 (Fig. 11). Cardiomyocyte contractility was higher (Fig. 12).

Figure 9. Endothelial uptake of fluorescein-encapsulated small unilamellar lipid vesicles within 5-10 minutes.

Figure 9

Endothelial uptake of fluorescein-encapsulated small unilamellar lipid vesicles within 5-10 minutes.

Figure 10. Comparison of ATP and total energy phosphate (TEP) in cardiomyocytes under chemical hypoxia indicates ATP-vesicles increase ATP and TEP by ∼2 fold (p < 0.

Figure 10

Comparison of ATP and total energy phosphate (TEP) in cardiomyocytes under chemical hypoxia indicates ATP-vesicles increase ATP and TEP by ∼2 fold (p < 0.05). Rat cardiomyocytes were incubated with KCN (5 mM) for a period of 30 minutes. (more...)

Figure 11. Redox status of cardiomyocytes under chemical hypoxia is increased by ATP delivery.

Figure 11

Redox status of cardiomyocytes under chemical hypoxia is increased by ATP delivery. Rat cardiomyocytes were incubated in either media or media + KCN (5 mM). After 30 minutes, the redox status of the cells was determined using Alamar Blue. ATP-vesicles (more...)

Figure 12. Cardiomyocyte contractility is maintained under chemical hypoxia by ATP-vesicles.

Figure 12

Cardiomyocyte contractility is maintained under chemical hypoxia by ATP-vesicles. The cells were stimulated with 0.5-4 Hz, 8-volt electric stimulator (MyoPacer™) and contractile velocity and duration of contraction were recorded after a 30-minute (more...)

During the process, we also have resolved several problems that were expected when highly fusogenic lipid vesicles are used. One problem was the stability of these vesicles. Although it has been proven that small unilamellar lipid vesicles are more stable in blood circulation,272 it is possible that these vesicles may fuse with each other when left alone for a long period of time.273 We have used a freeze-dry technique to keep these vesicles stable without particle size change during storage. Another problem was the temperature effect. All lipids have a phase transition temperature that will affect the fusion speed.271 , 274 Our current vesicles are created well above the liquid-crystalline-to-gel phase transition, circumventing the problem of lipid phase separation. Efforts are being made to further improve the delivery efficiency. Further research work may prove to be fruitful.

Other Pharmacologic Interventions

Drugs that can provide a physical environment to enhance the hypothermic effect or provide chemical protection, either directly or indirectly, have also been used in organ preservation with some success. So far, none of the drugs is dramatically effective in itself, but they have been reported as beneficial when used as adjuncts to enhance the quality of storage. Some of these drugs are listed below. Agents for specific organs, such as high potassium in cardioplegia and surfactant for lung preservation,275 will not be discussed here.

Oxygen Free Radical Scavengers

These chemicals are covered in chapter by Suzuki et al.

Prostaglandins

Prostaglandin E1 ( PGE1), prostacyclin (PGI2) and their analogues have been used in hypothermic preservation. Recognized to have a pulmonary vasodilatory effect to relieve vasoconstriction, they allow uniform distribution of perfusate throughout the lungs. They may also be associated with a number of other beneficial effects, such as inhibition of platelet aggregation, thrombus formation, neutrophil sequestration and lysosomal release. They are used before pulmonary artery flushing and improved preservation in lung and heart-lung transplantation.276 , 277 They may also have immunosuppressive and cytoprotective effects and may reduce vascular permeability.164 Protective effect for hypothermic liver storage has also been reported.278 However, the value and mechanism of prostaglandins in lung preservation are still controversial and its beneficial effect has been questioned.166 , 279

Calcium-Channel Blockers

Normally, the extracellular free calcium concentration is approximately 10−3 M, whereas the cytosolic free calcium concentration is about 10−7 M.280 Calcium influx into cells after reperfusion has been implicated in ischemic damage in a variety of organs and ischemic myocardium accumulates large amounts of calcium during reperfusion.280 Calcium also accumulates in hepatocytes and renal tubular cells that have been damaged by hypoxia and toxins.82 Agents blocking calcium flux may prove to be beneficial in preventing lung injury after reperfusion.166 Calcium-channel blockers like verapamil, nifedipine and nisoldipine have shown protective effect in hypothermic organ preservation.281 , 282 They are also believed to protect the heart from oxygen and substrate deprivation by decreasing the contractile activity and the energy demand.283 , 284

Na+/H+ Exchanger Inhibitors

Na+/H+ exchanger (NHE) inhibitors have received much attention in recent years. As stated above, intracellular Ca++ overload during ischemia and reperfusion is an important pathophysiological factor contributing to reduced post-ischemic recovery of cellular function. This is especially important in the myocardium. Although NHE activation is essential for the restoration of physiological pH, hyperactivation of NHE leads to a dramatic increase in intracellular Na+ concentration, which subsequently causes a marked increase in intracellular Ca++ concentration through the Na+/Ca++ exchanger.285 To date, eight isoforms of NHE have been identified. NHE-1 is the most predominant isoform expressed in the heart. The use of NHE-1 inhibitors such as amiloride, eniporide, cariporide, HOE-694, EMD-85131 and many bicyclic guanidines, has been shown to provide significant protection in models of myocardial ischemia and reperfusion, with consistent improvement in functional recovery, metabolic status, attenuation of arrhythmias, preservation of cellular ultrastructure and inhibition of apoptosis.286 - 288 However, the final role of these drugs has not been confirmed both in experimental studies or clinical trials.287 , 289

Platelet-Activating Factor (PAF) Antagonists

PAF binds to a specific site on platelets and is a potent inducer of platelet activation and aggregation. Activated platelets are capable of releasing potent mediators of inflammation.290 - 292 Platelet-activating factor antagonists, such as WEB2056, BN52021, E-5880 and CV-3988, have been reported to have beneficial effects during tissue ischemia, lung injury and liver preservation.293 - 295

Inhibition of Complement Activation

Complement activation has been implicated in the process of ischemia-reperfusion damage. Inhibition of this process may prove beneficial in organ preservation. Preliminary data using the complement inhibitors have shown promising results in lung and liver preservation,296 , 297 but controversy still exists.298

Nitric Oxide (NO)

One of the most extraordinary discoveries in modern medicine has been the finding that nitric oxide (NO) release, which accounts for the biological activity of the so-called endothelium-derived relaxing factor.299 With its widespread distribution in tissues and its ability to react with a range of molecules in the organism, a review appears incomplete without mentioning it. NO was Science magazine's “molecule of the year” in 1992 and its research has entered into almost all areas of biology and medicine, including organ preservation. NO donors such as nitrite, nitroglycerin, S-nitrosoglutathione, nitroprusside, or inhaled NO have been reported to be protective in hypothermic organ preservation, especially in lungs and liver.300 - 302 However, NO appears to be a double-edged sword that can be protective or destructive on any tissue or organ under different conditions. It is not surprising to see the opposite effect of NO in tissues and organs.303 , 304 It has become clear that, in some conditions, there may be too little NO, whereas in others there may be too much. “No one thing does only one thing” is probably more suitable for NO than for any other chemical. It is generally agreed that constitutively generated NO maintains microcirculation and endothelial integrity in many organs, while inducible NO synthase (iNOS)-governed NO production can be either beneficial or detrimental.305 The result of NO is affected by many other factors, such as the type of isoforms, doses, administering routes, existence of other mediators and, especially, other oxidants. There are many excellent reviews of this mediator306 , 307 and the use and controversy surrounding it will certainly continue.

Polyethylene Glycol (PEG)

The use of PEG in preservation solutions has been shown to be cytoprotective with improved organ function. It has also been shown to reduce rejection in clinical and experimental transplantations. These results have been shown in kidney,308 heart309 and liver.310 Several possible mechanisms of PEG have been proposed such as immunosuppression, prevention of osmotic swelling, reduction of lipid peroxidation and improvement of tissue ATP content.311 , 312 PEG is frequently incorporated into solutions for organ preservation.

Trimetazidine

Trimetazidine may be related to the restoration of ATP and CP, improvement of mitochondrial function, improved cellular resistance to hypoxic stress and antioxidant activity313 , 314 The use of trimetazidine has shown to be protective in kidney and heart preservations.315 , 316

Vasoactive Drugs

Propranolol can depress myocardial contractility and heart rate, thus exerting an energy-sparing effect on ischemic hearts. Propranolol can also suppress calcium accumulation during reperfusion.317 Studies of heart preservation have shown positive protective results.318 Other vasoactive drugs such as vasodilators319 , 320 and furosemide321 , 322 have also shown some effect.

Prevention of Cell Swelling

A. Mannitol

Mannitol is one of the most popularly used chemicals for reducing cell swelling. Mannitol has been shown to improve renal function when given before warm ischemia.323 Elevation of osmolality by mannitol increases collateral blood flow to the ischemic region through at least two mechanisms: (1) increasing osmolality results in the dilation of large arterial conductance vessels; and (2) producing an effect on the coronary circulation at a microvascular level, which increases collateral blood flow to ischemic regions.324

B. High-Molecular Impermeables

Due to their high molecular weight, these impermeables are used to maintain fluid osmolarity Both lactobionate and raffinose are important compounds in UW solution because of their large molecular mass.185 , 325

Membrane-Stabilizing Agents

Trehalose has the ability to stabilize the cell membrane structure by binding to phospholipid molecules in the bilayer. Trehalose is incorporated between the polar head groups of the phospholipids, thereby maintaining a specific distance between the molecules, which inhibits gelatinization and subsequent dysfunction of the bilayer under stressful conditions. It is one composition of a solution developed by Kyoto University in Japan.326 - 328

Steroids

Steroids are widely used as an adjunct to preservation, whether in the perfusate or by pretreatment of the donor and treatment of the recipient before reperfusion. The true mechanism of steroids is probably more complex, but their general anti-infammatory and membrane-stabilizing properties provide a protective role during organ preservation.115 , 329

Inhibition of Apoptosis (Programmed Cell Death)

Cells are eliminated in a variety of physiological settings by apoptosis, a genetically encoded process of cellular suicide. Apoptosis occurs as a consequence of global organ ischemia during isolation and storage before transplantation and various anti-apoptotic compounds have shown beneficial effects.330 Our group also tested a compound comprised mainly of phospholipids extracted from soybean in rabbit heart and rat liver preservations and obtained some very promising results.331 , 332

Other Metabolic Inhibitors

A. Chlorpromazine

The effect of chlorpromazine in protecting cells against ischemia is believed to be related to 2 mechanisms: (1) its pharmacological α-receptor blocking effect and its consequent vasodilating properties; 333 and (2) its membrane stabilization effect caused by inhibition of endogenous phospholipases, thus inhibiting the breakdown of the phospholipids.86 , 121

B. Butanedione monoxime (BDM)

Contracture of the myocardium is a serious event during ischemia and is thought to be related to depletion of ATP supplies.334 Butanedione monoxime (BDM) causes a marked depression of myocardial contractility with few side effects.335 It is also a vasodilator and can depress mobilization of sarcoplasmic reticular Ca++. It has been shown to enhance hypothermic heart preservation.334 , 336

Preconditioning

The earliest study of preconditioning was probably performed more than 4 decades ago by Dahl and Balfour.337 They found that the ischemic survival of rats was extended by placing them in nitrogen for a short period of time, letting them recover and then inducing anoxia. They also found that concentrations of lactate, pyruvate and ATP in the brain were higher during anoxia in the group subjected to pre-anoxia. When glycolysis was inhibited by iodoacetate, the pre-exposed advantage disappeared. Dahl and Balfour believed that the preconditioning was related to a faster production of ATP during anoxia because the first anoxia resulted in higher pyruvate and lactate reserves.

The phenomenon of preconditioning was described in 1986 by Murry et al,338 who discovered the protective effect of a brief ischemic period with regard to the detrimental consequences of subsequent prolonged ischemia. Since then, preconditioning has been shown to limit infarct size and to reduce ventricular arrhythmias during sustained ischemia and reperfusion. Brief repetitive periods of ischemia have been shown to retard cardiac energy metabolism during sustained ischemia in various animal experiments. This inhibition leads to sparing of high-energy phosphates and improves the time-averaged energy state during ischemia.339 , 340

Other Important Factors in Organ Preservation

Equilibration

To obtain optimal protective effects, the vascular and extracellular spaces have to be equilibrated with preservation fluid. This process varies for different organs and both flushing time and the volume of flushing fluid are important. For the kidney, fluid should also fill the tubular space. Only by a complete equilibration of the whole extracellular space of the kidney will the desired protection principles of buffering the “intracellular-like” composition be realized. Blood should be cleared from the preserved organs because (1) the trapped erythrocytes become less flexible as their ATP reserves are consumed and they tend to block the microcirculation after revascularization, causing ischemic damage;341 (2) the residual platelets can aggregate when temperature decreases;342 , 343 and (3) blood remaining in the transplanted organ may have undesirable immunological consequences.344 Cleaning the organs totally is almost impossible because washing out these blood cells can take as long as 10 hours.121 The equilibration of the different compartments of the kidney is nearly accomplished after 10 to 12 minutes of perfusion with a hydrostatic pressure of 120 cm H2O. The perfusion volume should be sufficient and that required by the kidney is at least 10 times the organ's weight, according to measurements of fluid substrates of the venous and tubular outflow.345 , 346 The heart needs 2 to 3 times its weight and the liver, 6 times its weight.346

Buffering

Buffering of the compartments of an organ is important because H+ ions and lactate can pass the cell membrane in anaerobiosis, a process that is pH-dependent. Since anaerobic glycolysis is the only energy source during anaerobiosis, this process is important for structural integrity. This issue has not been studied sufficiently. Using a rat model, Shiraishi et al347 demonstrated that lungs treated at pH 7.75 showed a significantly lower pulmonary artery pressure and wet-to-dry weight ratio than those treated at pH 7.26 or 7.96. Experimental data and clinical studies show that a pH value of 6.2 at 35°C or 6.6 at 5°C is marginally tolerated. Intrarenal acidosis beyond these values appears to be the first limiting factor for graft function in kidneys.348 , 349

Nature Still Provides the Best Preservation

While various attempts have been made to further extend cold preservation time for solid organs, it is disappointing to realize that decades of research studies have not extended preservation times for the heart and lungs, even for a few hours. It has been shown repeatedly and is also reflected in this book, that using perfusion can extend survival time. This is obvious because perfusion can supply oxygen and nutrients continuously and remove metabolic wastes—all are critical to organ survival. There are complicated issues regarding using the perfusion technique. For one thing, unlike single-flush hypothermic preservation that needs little attention during the preservation period, mechanical perfusion is labor intensive and expensive. It is difficult to gain acceptance in the managed care era. Perfusion itself can pose additional risks to the organs that are not present in hypothermic storage, such as damage to vascular endothelial cells, the possibility of mechanical failure and particle formation in the perfusates.92 , 350 To overcome these problems, a simple heart-lung autoperfusion was developed that did provide longer preservation time for the heart, but not the lungs.351 - 355 We tried this technique and found one possible reason for lung damage: the circulating aggregates from blood-air (and possible artificial circuit) interface.356 We eliminated the blood-air interface and expanded the organ block to include the heart, lungs, liver, pancreas, duodenum and both kidneys. In that preparation, the heart pumps blood, the lungs oxygenate the blood, the liver processes normal chemical reactions and metabolic wastes are removed by the kidneys. The organs are self-contained and a respirator is the only artificial equipment needed for its survival. The organs could be preserved for more than 2 days with good function.146 , 147 , 357 In one study, using 6 pairs of adult mongrel dogs, the organs were preserved from 24 to 33 hours (mean 26.7 ± 1.4 hours) in the multiple organ block along with intermittent infusion of plasma from hibernating animals. Orthotopic lung transplantation was performed and the dogs were observed for 24 hours with good lung function.149 Figure 13 is an example of the lungs at 30 hours of preservation. These lungs show the best quality of any preservation technique we have seen.

Figure 13. An example of the lungs at 30 hours of preservation.

Figure 13

An example of the lungs at 30 hours of preservation. Not only do the lungs look normal, tissue wet/dry ratio was also normal.

Metabolic management during perfusion is not the main focus in this chapter because it requires totally different strategies. However, the above example indicates that physiologically, using an animal's own heart as a pump and its own blood as a perfusion medium, provides the simplest and possibly the best environment for long-term organ survival. The normothermic autoperfusion technique was used early in the history of organ preservation. Due to reasons similar to mechanical perfusion, it is probably difficult to gain acceptance in clinical practice. Because of the technical difficulties in the setup and the monitoring processes, even the simplest autoperfusion was used only sporadically.292 , 358 , 359 We do not know the other possible mechanisms of the plasma from hibernating animals in this preparation, except its obvious role in reducing liver congestion.146 , 147 , 357 The above example appears to show that using natural circulation is still the best option for long-term organ preservation.

Future Perspective

Over the past 40 years, slow progress has been made in organ preservation for transplantation. Despite the extension of preservation times for the kidneys and livers, safe preservation times for the heart and lungs are still very short.360 In the past decade, there has been a gradual expansion of marginal donor organs used as a result of a more careful selection of solutions for individual organs,361 , 362 but there has not been a dramatic extension in preservation times.363 Longer ischemic times are often associated with early heart failure and chronic fibrosis after transplantation.364 - 366 Many of the compounds mentioned in this chapter, albeit with great theoretical advantages and promising experimental results, have not made their way into clinical practice. This is mainly because sustained protective results are lacking in a larger number of animal experiments and/or clinical trials. Among the reasons for this is the period of organ ischemia outside the body, which is subject to a number of biochemical stress factors that cause a combined-damage effect that cannot be prevented or treated by a single drug. Another reason is that our knowledge of known critical components of tissue damage is still incomplete. For example, although the decline of energy production plays a major role in tissue ischemic damage and cell death is well recognized, the critical point at which cells die is far from settled. Evidence shows that it is very unlikely that there is a single critical level of ATP for all tissues and organs. Even in the same organ, this critical level may be different due to the existence of other factors. As such, it is not surprising to realize the controversy toward the relationship between high-energy phosphate concentration and organ function and viability. In the literature, both positive54 , 367 , 368 and negative72 , 81 relationships have been reported. There is also the possibility of ATP compartmentation in subcellular organelles. Our current techniques can only determine the total tissue content of a particular metabolite and may not provide a good estimate of its concentration in a specific subcellular compartment. If a large concentration gradient of any compound across the mitochondrial membrane is present, the interpretation will be difficult.369 Furthermore, it is not the sole concentration of ATP that is critical to cell survival, but it is the rate of ATP turnover that is more important to the survival of the cells.370 This will gradually change when our knowledge of biochemical and immunological mechanisms of organ damage improves.

Further improvement of preservation techniques is critical with the increased use of marginal and suboptimal donor organs and organs from nonheart-beating donors (NHBD). However, organ preservation is only one ring in the chain of organ damage, beginning before retrieval and ending at restoration of blood supply. Many of the organs from NHBD are severely damaged during the long period of hypotension before retrieval, making preservation more critical.

True long-term organ preservation for months or years would be a valuable future development. Such techniques would transform transplantation into elective surgery, probably eliminate recipient waiting lists, make the process of time-consuming matching of donors and recipients more possible and provide time for immunological pretreatment of the recipients or donor organs.371 It is unlikely that the current hypothermic storage or mechanical perfusion techniques will achieve this goal. However, hypothermic storage is simple and the most cost-effective. A gradual extension of current hypothermic preservation time will be possible when there is more understanding of the specific metabolic demands and physiologic requirements of different cells and organs. This would include the nature of hypoxic damage to cells and organs and the methods that avoid or reduce various kinds of damage. It may require a combined treatment of several drug categories to offset injuries caused by different mechanisms,60 , 372 , 373 and the treatment is probably organ-specific.374

Acknowledgements

This study was supported in part by NIH grants HL64186, DK74566 and AR52984. The author wishes to thank Drs. Dongping Hua, Benjamin Chiang and William Ehringer for their experimental contributions; Jiusheng Ye for his HPLC work and Margaret A. Abby for her editorial assistance.

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