Maintenance Of Adenosine Triphosphate Research Articles

Oxygenated versus non-oxygenated flush out during deceased donor liver procurement: The first proof-of-concept study in humans.

Liver transplantation is used for treating end-stage liver disease, fulminant hepatitis, and oncological malignancies and organ shortage is a major limiting factor worldwide. The use of grafts based on extended donor criteria have become internationally accepted. Oxygenated machine perfusion technologies are the most recent advances in organ transplantation; however, it is only applied after a period of cold ischemia. Due to its high cost, we aimed to use a novel device, OxyFlush®, based on oxygenation of the preservation solution, applied during liver procurement targeting the maintenance of ATP during static cold storage (SCS). Twenty patients were randomly assigned to the OxyFlush or control group based on a 1:1 ratio. In the OxyFlush group, the perfusion solution was oxygenated with OxyFlush® device while the control group received a non-oxygenated solution. Liver and the common bile duct (CBD) biopsies were obtained at three different time points. The first was at the beginning of the procedure, the second during organ preparation, and the third after total liver reperfusion. Biopsies were analyzed, and adenosine triphosphate (ATP) levels and histological scores of the liver parenchyma and CBD were assessed. Postoperative laboratory tests were performed. OxyFlush® was able to maintain ATP levels during SCS and improved the damage caused by the lack of oxygen in the CBD. However, OxyFlush® did not affect laboratory test results and histological findings of the parenchyma. We present a novel low-cost device that is feasible and could represent a valuable tool in organ preservation during SCS.

Closed system processing variables affect post-thaw quality characteristics of cryopreserved red cell concentrates.

Differences in manufacturing conditions using the Haemonetics ACP 215 cell processor result in cryopreserved red cell concentrates (RCCs) of varying quality. This work studied the effect of processing method, additive solution, and storage duration on RCC quality to identify an optimal protocol for the manufacture of cryopreserved RCCs. RCCs were pooled-and-split and stored for 7, 14, or 21 days before cryopreservation. Units were glycerolized with the ACP 215 using a single or double centrifugation method. After thawing, the RCCs were deglycerolized, suspended in AS-3, SAGM, ESOL, or SOLX/AS-7, and stored for 0, 3, 7, 14, or 21 days before quality testing. Quality assessments included hemoglobin content, hematocrit, hemolysis, adenosine triphosphate (ATP), supernatant potassium, and mean cell volume. Both glycerolization methods produced RCCs that met regulatory standards for blood quality. Dual centrifugation resulted in higher hemoglobin content, fewer processing alerts, and a shorter deglycerolization time than single centrifugation processing. Units processed with AS-3 and ESOL met regulatory standards when stored for up to 21 days pre-cryopreservation and 21 days post-deglycerolization. However, ESOL demonstrated superior maintenance of ATP over RBCs in AS-3. Some RCCs suspended in SAGM and SOLX exceeded acceptable hemolysis values after 7 days of post-deglycerolization storage regardless of pre-processing storage length. When manufacturing cryopreserved RCCs using the ACP 215, dual centrifugation processing with AS-3 or ESOL additive solutions is preferred, with storage periods of up to 21 days both pre-processing and post-deglycerolization.

Myocardial Adaptation in Pseudohypoxia: Signaling and Regulation of mPTP via Mitochondrial Connexin 43 and Cardiolipin.

Therapies intended to mitigate cardiovascular complications cannot be applied in practice without detailed knowledge of molecular mechanisms. Mitochondria, as the end-effector of cardioprotection, represent one of the possible therapeutic approaches. The present review provides an overview of factors affecting the regulation processes of mitochondria at the level of mitochondrial permeability transition pores (mPTP) resulting in comprehensive myocardial protection. The regulation of mPTP seems to be an important part of the mechanisms for maintaining the energy equilibrium of the heart under pathological conditions. Mitochondrial connexin 43 is involved in the regulation process by inhibition of mPTP opening. These individual cardioprotective mechanisms can be interconnected in the process of mitochondrial oxidative phosphorylation resulting in the maintenance of adenosine triphosphate (ATP) production. In this context, the degree of mitochondrial membrane fluidity appears to be a key factor in the preservation of ATP synthase rotation required for ATP formation. Moreover, changes in the composition of the cardiolipin’s structure in the mitochondrial membrane can significantly affect the energy system under unfavorable conditions. This review aims to elucidate functional and structural changes of cardiac mitochondria subjected to preconditioning, with an emphasis on signaling pathways leading to mitochondrial energy maintenance during partial oxygen deprivation.

Adenosine triphosphate maintenance by branched chain amino acids as a novel neuroprotective strategy for retinal neurodegenerative diseases.

Adenosine triphosphate maintenance by branched chain amino acids as a novel neuroprotective strategy for retinal neurodegenerative diseases.

Mannose and fructose metabolism in red blood cells during cold storage in SAGM.

Alternate sugar metabolism during red blood cell (RBC) storage is not well understood. Here we report fructose and mannose metabolism in RBCs during cold storage in SAGM and the impact that these monosaccharides have on metabolic biomarkers of RBC storage lesion. RBCs were stored in SAGM containing uniformly labeled 13 C-fructose or 13 C-mannose at 9 or 18 mmol/L concentration for 25 days. RBCs and media were sampled at 14 time points during storage and analyzed using ultraperformance liquid chromatography-mass spectrometry. Blood banking quality assurance measurements were performed. Red blood cells incorporated fructose and mannose during cold storage in the presence of glucose. Mannose was metabolized in preference to glucose via glycolysis. Fructose lowered adenosine triphosphate (ATP) levels and contributed little to ATP maintenance when added to SAGM. Both monosaccharides form the advanced glycation end product glycerate. Mannose activates enzymes in the RBC that take part in glycan synthesis. Fructose or mannose addition to RBC SAGM concentrates may not offset the shift in metabolism of RBCs that occurs after 10 days of storage. Fructose and mannose metabolism at 4°C in SAGM reflects their metabolism at physiologic temperature. Glycerate excretion is a measure of protein deglycosylation activity in stored RBCs. No cytoprotective effect was observed upon the addition of either fructose or mannose to SAGM.

CO2 -dependent metabolic modulation in red blood cells stored under anaerobic conditions.

Anaerobic red blood cell (RBC) storage reduces oxidative damage, maintains adenosine triphosphate (ATP) and 2,3-diphosphoglycerate (DPG) levels, and has superior 24-hour recovery at 6 weeks compared to standard storage. This study will determine if removal of CO2 during O2 depletion by gas exchange may affect RBCs during anaerobic storage. This is a matched three-arm study (n = 14): control, O2 and CO2 depleted with Ar (AN), and O2 depleted with 95%Ar/5%CO2 (AN[CO2 ]). RBCs in additives AS-3 or OFAS-3 were evenly divided into three bags, and anaerobic conditions were established by gas exchange. Bags were stored at 1 to 6°C in closed chambers under anaerobic conditions or ambient air, sampled weekly for up to 9 weeks for a panel of in vitro tests. A full metabolomics screening was conducted for the first 4 weeks of storage. Purging with Ar (AN) results in alkalization of the RBC and increased glucose consumption. The addition of 5% CO2 to the purging gas prevented CO2 loss with an equivalent starting and final pH and lactate to control bags (p > 0.5, Days 0-21). ATP levels are higher in AN[CO2 ] (p < 0.0001). DPG was maintained beyond 2 weeks in the AN arm (p < 0.0001). Surprisingly, DPG was lost at the same rate in both control and AN[CO2 ] arms (p = 0.6). Maintenance of ATP in the AN[CO2 ] arm demonstrates that ATP production is not solely a function of the pH effect on glycolysis. CO2 in anaerobic storage prevented the maintenance of DPG, and DPG production appears to be pH dependent. CO2 as well as O2 depletion provides metabolic advantage for stored RBCs.

Age-related alteration of intracellular ATP maintenance in the cell suspensions of mice cerebral cortex

Neurological alteration in the aging brain has been suggested to be due to, in part, a declined cellular energy metabolism. In order to understand age-related alteration of intracellular ATP maintenance, the present in vitro study measured change of intracellular adenosine triphosphate (ATP) content in cell suspensions of cerebral cortex isolated from male ICR mice aged 2 days (infant), 8 weeks (young adult) and 12 months (aged) under several different conditions, using the chemiluminescence technique. Among the three different ages, significant decrease of intracellular ATP content by oxygen deprivation for 15 min was observed in the cell suspensions of cerebral cortex from 12-month-old mice ( P<0.05). When cell suspensions of 8-week cerebral cortex were incubated with or without glucose (0–60 min), intracellular ATP content decreased in a time-dependent manner under both conditions, but depletion rate was significantly high in the glucose-free condition. This decrease was maximally restored by adding 1 mM glucose as tested. In addition, the ability for intracellular ATP maintenance in the presence or absence of glucose was age-dependently different. The rank order of difference of intracellular ATP content between with and without glucose was 3 months>12 months>2 days. The highest decrease of intracellular ATP content by incubation without glucose was observed in the 12-month samples. Sodium cyanide (100 μM) produced a gradual ATP depletion in cerebral cortex suspended from 2-day-old mice, but rapid change in both 8-week and 12-month samples. Combination of cyanide and iodoacetate (3.5 mM) rapidly depleted the intracellular ATP content in all age groups tested. These results suggest that the aging process in the cerebral cortex of mice is accompanied by alteration of maintenance of intracellular ATP homeostasis under a given condition, and this may be associated with pathological change of overall mechanisms involved in the development of neuronal disease in the senescent brain.

Glucose-6-phosphatase mRNA levels in kidney isolated tubule suspensions are increased by dexamethasone and decreased by insulin

The strong induction of renal glucose-6-phosphatase (G6Pase) during starvation has been suggested to be responsible for the increased role of the kidney in glucose production during long-term fasting. To investigate whether this induction may be caused by a direct hormonal effect on the renal proximal tubular cell, we incubated rat renal tubule suspensions in the presence of glucocorticoids or insulin for 6 hours; normoxia was required, since hypoxic conditions were associated with markedly decreased G6Pase mRNA levels despite maintenance of adenosine triphosphate (ATP) levels. The G6Pase mRNA level was increased twofold to threefold by 10 −8 to 10 −5 mol/L dexamethasone (DXM), whereas the most effective concentration of insulin, 10 −9 mol/L, induced only a 40% decrease. These results suggest that the increased role of the kidney in glucose production during long-term starvation could be linked to a direct effect of glucocorticoids on renal G6Pase.

Insulin pretreatment protects the liver from ischemic damage during Pringle's maneuver

Although maintenance of adenosine triphosphate (ATP) levels is important to restore liver functions during anoxia, ATP production by oxidative phosphorylation is inhibited during Pringle's maneuver, and only a little ATP can be supplied by glycolysis. The glycolytic activity of the liver is controlled by the nutritional condition and hormones. Enhancement of glycolytic activity by insulin may increase ATP production and thus may protect the liver from ischemia. Rats were divided into three groups: fasted group, food was withheld for 24 hours; fed group, food was provided ad libitum; and insulin group, fed rats were administered insulin (12 units/kg during a 30-minute period) before portal triad clamping (PTC) was performed. After laparotomy was performed, PTC was performed for 30 minutes. The fructose 2,6-bisphosphate (F-2,6-BP) level, the hepatic levels of lactate and ATP, the bile flow rate, the plasma levels of aspartate transaminase and lactate dehydrogenase, and the indocyanine green clearance were measured at appropriate times. The hepatic F-2,6-BP levels before PTC in the fasted, fed, and insulin groups were 6.2 +/- 3.8, 55.6 +/- 10.6, and 122.2 +/- 31.3 nmol/gm dry weight liver, respectively. The glycolytic activity of the insulin group before PTC was significantly enhanced compared with that of the other groups. Lactate was more rapidly accumulated in livers of the insulin group during PTC than in those of the other groups. The ATP level and energy charge during PTC of the insulin group were higher than those of the other groups. The bile flow rate and indocyanine green clearance after PTC were restored in the order of the insulin, fed, and fasted groups. Insulin administration before PTC increased the hepatic F-2,6-BP content and enhanced glycolytic activity. Insulin pretreatment combined with feeding improved the hepatic energy metabolism during PTC and restored the liver functions after PTC. Insulin has protective effects on the liver during PTC.

Effect of cerebral blood flow generated during cardiopulmonary resuscitation in dogs on maintenance versus recovery of ATP and pH.

Cardiopulmonary resuscitation with external chest compression generates low perfusion pressures that may be inadequate for restoring cerebral metabolism and may worsen intracellular pH. We tested the hypothesis that cerebral reperfusion with a low perfusion pressure after arrest restores brain adenosine triphosphate (ATP) and pH to levels attained at the same perfusion pressure without preceding complete ischemia. Brain ATP and intracellular pH were measured by magnetic resonance spectroscopy, and cerebral blood flow was measured with microspheres in anesthetized dogs. External chest compressions were begun in group A (n = 6) immediately after the onset of arrest (ie, arrest time zero) and in group B (n = 10) after 6 minutes of arrest (ie, arrest time 6 minutes). In both groups, mean cerebral perfusion pressure was regulated at 30 mm Hg for 70 minutes by adjustment of inflation pressure of a pneumatic thoracic vest. At 12 minutes of resuscitation, cerebral blood flow was 27 +/- 4 mL/min per 100 g in group A and 21 +/- 4 mL/min per 100 g in group B, but ATP in group B (58 +/- 10% of prearrest) was less than in group A (105 +/- 6%). With prolonged resuscitation, ATP deteriorated to near zero levels in dogs in group B, with blood flow less than 15 mL/min per 100 g. Dogs with greater blood flow never achieved complete metabolic recovery. In group B, intracellular pH was unchanged from the 6.3 value at the start of resuscitation, even in those dogs with extremely low blood flows. Levels of cerebral perfusion pressure sufficient to maintain cerebral oxidative metabolism without complete ischemia during cardiopulmonary resuscitation are not sufficient to restore metabolism after complete ischemia during cardiopulmonary resuscitation. However, low "trickle" blood flow did not worsen intracellular acidosis.

Reactive oxygen species and human spermatozoa.

Abstract: Under moderate conditions, reactive oxygen species (ROS) have been shown to inhibit sperm motility after several hours of incubation. The rapid decrease in flagellar beat frequency observed within the first hour of contact between ROS and spermatozoa was associated with a rapid loss of intracellular adenosine triphosphate (ATP). Motility of intact spermatozoa ceased when their ATP concentration was reduced by 85 ±‐ 5%. Axonemal damage was confirmed when ROS‐treated spermatozoa could not reactivate motility after demembranation in a medium containing magnesium adenosine triphosphate (Mg.ATP). However, in conditions allowing rephosphorylation of the axonemes (addition of cyclic adenosine monophosphate, or cAMP, and protein kinase or sperm extracts to the demembranation medium), the motility could reactivate. Three lines of evidence suggested that ATP depletion induced by ROS treatment was responsible for the effects observed in spermatozoa. First, the rapid decrease in intracellular ATP observed after ROS treatment was closely followed by a decrease in beat frequency, loss of intact sperm motility, and axonemal damage due to insufficient phosphorylation. Second, incubation of spermatozoa with the combination pyruvate‐lactate allowed maintenance of sperm ATP at a normal level and prevented the effects of ROS; furthermore, spermatozoa immobilized after ROS treatment, then supplemented with pyruvate‐lactate, were able to reinitiate motility in parallel with an increase in their ATP level. Third, treatment of spermatozoa with rotenone, an ATP depleting agent, produced effects similar to ROS treatment and could also be reversed by the addition of pyruvate‐lactate. These data are consistent with the conclusion that ROS treatment produced axonemal damage mostly as a result of ATP depletion However, other mechanisms, such as inhibition of sperm protein kinases, are probably involved.

Relationship between ATP resynthesis and calcium accumulation in the reperfused rat heart.

1. The postulate that the composition of solutions used to reperfuse ischaemic hearts may modulate their ability to synthesize high-energy compounds was tested in isolated rat hearts subjected to 30 min normothermic ischaemia and then reperfused with either Krebs'-Henseleit buffer (K-H) for 20 min (control reperfusion, CR), or a 'myocardial protective solution' (MPS) for 5 min, followed by 15 min K-H (modified reperfusion, MR). The 'myocardial protective solution' was designed to protect against damage caused by sodium and calcium accumulation and by free radicals. Metabolic precursors were also included to promote and support adenosine triphosphate (ATP) resynthesis during reperfusion under both aerobic and hypoxic conditions. 2. 31P nuclear magnetic resonance (NMR) was used to measure tissue ATP and creatine phosphate (CP), and atomic absorption spectrometry was used to measure Ca++. Early during CR, ATP recovered to 28% of the pre-ischaemic value, but fell to 5.5% with continued perfusion. Similarly, CP recovered to 45.5% of the pre-ischaemic level during early CR but fell to 25.5% with continued perfusion. 3. Better maintenance of ATP was seen during MR with oxygenated MPS (O2-MR), the final ATP remaining at 16.9% of the pre-ischaemic level. During O2-MR, CP recovered to 43.55 of the pre-ischaemic level but was not maintained and fell to a final level of 29.5%. 4. During MR with O2-free MPS (non-O2-MR), there was no reperfusion-associated fall in ATP or CP, with the levels maintained at 26.6% and 34.55, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies in Red Blood Cell Preservation 3. A Phosphate‐Ammonium‐Adenine Additive Solution1

The maintenance of adenosine triphosphate (ATP) in red blood cells (RBC) during storage is largely dependent on the integrity of glycolytic metabolism in the Embden-Meyerhof pathway. Meryman et al. [Transfusion 1986; 26:500-505] hypothesized that a solution that increased the surface tension of the corpuscles through hypotonic swelling might retard the development of echinocytosis and membrane loss by the shedding of exocytic vesicles. Unexpectedly, maintenance of good ATP levels and satisfactory RBC survivals were found for a long as 18 weeks. The purpose of our study was to test their observations and to explore the possible mechanisms. Equal parts of units of packed RBC were stored in the experimental preservative and, for comparison, in ADSOL. The most notable findings were ATP values at 4 weeks averaging 5.2 mumol/g Hb (130% of initial) and at 12 weeks 2.9 mumol/g Hb (73% of initial), whereas these values declined as expected in ADSOL. Mean RBC diameters and surface areas by morphometric analysis were not significantly different in the two preservatives indicating the absence of any hypotonic swelling. The morphology scores of the RBC were significantly better throughout (p less than 0.05) than in ADSOL. The shedding of exocytic hemoglobin-containing vesicles was essentially the same in both preservatives. Our data confirm the observation that ATP levels are well maintained for at least 12 weeks, but do not show any evidence that hypotonic swelling was a part of the mechanism.

Studies in Red Blood Cell Preservation - 3. A Phosphate-Ammonium-Adenine Additive Solution

The maintenance of adenosine triphosphate (ATP) in red blood cells (RBC) during storage is largely dependent on the integrity of glycolytic metabolism in the Embden-Meyerhof pathway. Meryman et al. [Transfusion 1986;26:500-505] hypothesized that a solution that increased the surface tension of the corpuscles through hypotonic swelling might retard the development of echinocytosis and membrane loss by the shedding of exocytic vesicles. Unexpectedly, maintenance of good ATP levels and satisfactory RBC survivals were found for as long as 18 weeks. The purpose of our study was to test their observations and to explore the possible mechanisms. Equal parts of units of packed RBC were stored in the experimental preservative and, for comparison, in ADSOL®. The most notable findings were ATP values at 4 weeks averaging 5.2 μmol/g Hb (130% of initial) and at 12 weeks 2.9 μmol/g Hb (73% of initial), whereas these values declined as expected in ADSOL. Mean RBC diameters and surface areas by morphometric analysis were not significantly different in the two preservatives indicating the absence of any hypotonic swelling. The morphology scores of the RBC were significantly better throughout (p<0.05) than in ADSOL. The shedding of exocytic hemoglobin-containing vesicles was essentially the same in both preservatives. Our data confirm the observation that ATP levels are well maintained for at least 12 weeks, but do not show any evidence that hypotonic swelling was a part of the mechanism.

Preservation of high-energy phosphates in human myocardium A phosphorus 31-nuclear magnetic resonance study of the effect of temperature on atrial appendages

After prolonged exposure to low temperatures (1 degree and 4 degrees C), human atrial trabeculae show poor recovery of contraction. At somewhat higher temperatures (12 degrees and 20 degrees C), recovery is much better (Keon and associates. Ann Thorac Surg 1988;46:337-41). Although better preservation of adenosine triphosphate and therefore improved contractile recovery might be expected after exposure to lower temperatures, it remained possible that, below a certain temperature, adenosine triphosphate-generating mechanisms could be slowed more than adenosine triphosphate utilization. To investigate this phenomenon further, we followed the time course of metabolic changes in human atrial appendages, harvested during cardiac bypass operations, at 1 degree, 4 degrees, 12 degrees, and 20 degrees C using high-resolution 31P and 1H nuclear magnetic resonance spectroscopy. The results are quantitated by correlation with data obtained from biochemical assays on quick-frozen tissues. Initial adenosine triphosphate levels in myocytes of human atrial appendages are 3.3 to 4.3 mumol.gm-1 tissue wet weight. At 20 degrees C, adenosine triphosphate disappears after 6 hours; at 12 degrees C, about half the initial adenosine triphosphate is still observable at this time; at 4 degrees C or 1 degree C, the decline is still slower. Only a small contribution toward adenosine triphosphate maintenance comes from creatine phosphate, since creatine phosphate, inorganic phosphate, and total creatine levels in the appendage are low (less than 2 mumol.gm-1 tissue wet weight). Glycolysis is active at all temperatures; the rate of glycolysis correlates positively with increasing temperature. Adenosine triphosphate generated by glycolysis falls just short of demand at all temperatures, but the difference is small at 1 degree and 4 degrees C. These studies lead us to conclude that the relatively poor recovery of contractile response of human atrial trabeculae, together with contracture reported previously at lower temperatures (1 degree and 4 degrees C), is not due to a failure to maintain adenosine triphosphate levels.

Protection of hippocampal slices from young rats against anoxic transmission damage is due to better maintenance of ATP.

1. Dentate granule cells in hippocampal slices from young rats (aged 30-40 days) are more resistant to damage from 10 min of anoxia than are granule cells from adult rats. The evoked population spike from these cells recovers to 78% of its pre-anoxic amplitude in young animals while in adult animals it shows only 4% recovery. This increased resistance is associated with higher levels of adenosine triphosphate (ATP) during the anoxic period. 2. When the duration of anoxia in slices from young animals is increased to 15 min, ATP falls to levels found in adult tissue after 10 min of anoxia. The dentate granule cells in slices from young animals show little recovery of the evoked response (19%) after such an exposure to anoxia. 3. When slices from young animals are subjected to 10 min of anoxia in low-glucose (2 mM) artificial cerebrospinal fluid, ATP levels fall to those found in adult tissue after 10 min of anoxia and the evoked response from the dentate granule cells again shows little recovery (10%). 4. The evoked response in the CA1 pyramidal cell layer of slices from young rats is more resistant to damage from 5 or 7 min anoxia than it is in slices from adults. Thus this region, also, shows an age-dependent increase in susceptibility to anoxic damage. ATP levels in the CA1 region of tissue from young animals at the end of 5 and 7 min anoxia are greater than ATP levels in tissue from adult animals after these same anoxic exposures. 5. Basal levels of 45Ca accumulation are greater in CA1 and dentate gyrus from young rats. However, the percentage increases during 10 min of anoxia are less than one-half the values in slices from adult animals. 6. The results suggest that the increased resistance of slices from young animals to anoxic transmission damage may be explained by the better maintenance of ATP in synaptic regions of these slices during anoxia. This may confer the increased resistance by lowering the anoxic increase in cell Ca2+.

Development of an optimized additive solution containing ascorbate-2-phosphate for the preservation of red cells with retention of 2,3 diphosphoglycerate.

An additive solution containing adenine, ascorbate-2-phosphate, sodium phosphate, dextrose, and saline was developed for packed red cell preservation. The combination of all components was simultaneously optimized so that the resulting solution produced the maximum retention of both red cell adenosine triphosphate (ATP) and 2,3 diphosphoglycerate (2,3 DPG) concentrations. Fourteen nutrient combinations were tested; each combination was evaluated for 42 days of storage using cells from three donors. The nutrient combinations were chosen with the aid of a computerized experimental design process. Results of the experiments were modeled by regression analysis, and the model was optimized to produce the "best" formulation for simultaneous maintenance of ATP and 2,3 DPG. The resulting mathematically optimal formulation was tested in the laboratory using 10 units of red cells. With this solution, it was possible to store red cells for 42 days with retention of 45 to 55 percent of the initial ATP and 85 to 150 percent of the initial 2,3 DPG. Red cell lysis was low (0.8 percent), and most of the cells were biconcave discs (by scanning electron microscopy) at the end of storage. The studies were carried out in an efficient manner by using computer-optimized experimental design techniques coupled with multiple regression modeling and subsequent computer optimization of the models. This experimental approach has potential application to many current blood banking procedures. This additive solution should maintain viable red cells for 42 days. In addition, the solution will maintain red cell 2,3 DPG throughout storage.

Disulfide-linked and transglutaminase-catalyzed protein assemblies in platelets

Energy depletion induces the formation of disulfide-linked and transglutaminase-catalyzed protein assemblies in platelets. The disulfide type polymers, formed following incubation at 37 degrees C in the absence of adenosine triphosphate (ATP)-generating precursors, are composed of cytoskeletal proteins and are associated with a decrease of reduced glutathione levels accompanying ATP depletion. The maintenance of ATP and reduced glutathione levels to, respectively, 34% and 47% of their original values is sufficient to prevent the formation of both polymer types. The transglutaminase-type cross-links are formed in the presence of calcium in either “energy-depleted” or thrombin stimulated platelets. 125I-surface-labeled membrane proteins, presumably transmembrane proteins, are incorporated into the transglutaminase- catalyzed cross-linked polymer of thrombin-stimulated platelets. Glycoproteins IIb and IIIa are not essential to the polymer formation, since thrombasthenic platelets treated with thrombin exhibit the same type of labeled polymer. The transglutaminase-catalyzed polymer formation following thrombin stimulation of platelets is inhibited by a calcium channel blocker, an intracellular calcium antagonist, as well as other inhibitors such as indomethacin, dibutyryl cyclic AMP, and prostaglandin E1. Although the evidence points to the formation of transglutaminase-catalyzed cross-linking in the cytoplasmic compartment, additional cross-linking of extruded components cannot be excluded.

Disulfide-Linked and Transglutaminase-Catalyzed Protein Assemblies in Platelets

Energy depletion induces the formation of disulfide-linked and transglutaminase-catalyzed protein assemblies in platelets. The disulfide type polymers, formed following incubation at 37 degrees C in the absence of adenosine triphosphate (ATP)-generating precursors, are composed of cytoskeletal proteins and are associated with a decrease of reduced glutathione levels accompanying ATP depletion. The maintenance of ATP and reduced glutathione levels to, respectively, 34% and 47% of their original values is sufficient to prevent the formation of both polymer types. The transglutaminase-type cross-links are formed in the presence of calcium in either "energy-depleted" or thrombin stimulated platelets. 125I-surface-labeled membrane proteins, presumably transmembrane proteins, are incorporated into the transglutaminase-catalyzed cross-linked polymer of thrombin-stimulated platelets. Glycoproteins IIb and IIIa are not essential to the polymer formation, since thrombasthenic platelets treated with thrombin exhibit the same type of labeled polymer. The transglutaminase-catalyzed polymer formation following thrombin stimulation of platelets is inhibited by a calcium channel blocker, an intracellular calcium antagonist, as well as other inhibitors such as indomethacin, dibutyryl cyclic AMP, and prostaglandin E1. Although the evidence points to the formation of transglutaminase-catalyzed cross-linking in the cytoplasmic compartment, additional cross-linking of extruded components cannot be excluded.

Advantages of blood cardioplegia over continuous coronary perfusion or intermittent ischemia

A safe, effective method of producing a quiet, bloodless heart for 2 hours or more has been developed experimentally and used clinically. Aliquots of oxygenated blood from the heart/lung machine are altered (calcium chelation, Ca ++ 0.6 mEq. per liter; alkalinization, pH 7.8; hyperkalemia, 30 mEq. per liter; and cooling, 22° C.) and infused by the perfusionist from the pump console into the proximal aortic root or coronary ostia (during aortic valve replacement) every 20 minutes. Of 17 dogs placed on cardiopulmonary bypass, seven received continuous coronary perfusion for 4 hours. In 10 dogs, the aorta was clamped for 2 hours (myocardial temperature 22° C), and 150 c.c. of pump blood was injected into the aortic root every 20 minutes. In five dogs this blood was unaltered (simulating intermittent release of the aortic clamp), and in five others the blood was modified with the cardioplegic additives. The best myocardial protection occurred with blood cardioplegia with, near-normal compliance (80 percent recovery*), normal left ventricular contractility, and maintenance of adenosine triphosphate (ATP). In contrast, with continuous coronary perfusion, ATP and contractility were preserved but compliance was reduced 60 percent.* With intermittent 22° C. ischemia, severe depression of ATP (42 percent*), compliance (83 percent*), and contractility (52 percent*) occurred. Next, the hospital course of 148 consecutive, nonrandomized patients was analyzed. In the 77 patients with continuous coronary perfusion or intermittent ischemia, there were 59 coronary artery bypass grafts (CABG), 15 aortic valve replacements (AVR), and three AVR plus CABG. In 71 patients undergoing prolonged aortic clamping (longest 124 minutes) with blood cardioplegia, there were 55 CABG, 11 AVR, and five AVR plus CABG. The average ischemic time was much longer in the blood cardioplegic group (43 ± 33 versus 19 ± 18 minutes, 2 S.D.). Despite longer ischemia, results with blood cardioplegia were superior in regard to postoperative CK (796 versus 1,251 units per liter*), SGOT (59 versus 99 units per liter*), infarct rate (ECG, 1/71 versus 11/77*), cardiac output (5.2 versus 3.9 L. per minute*), need for circulatory support (1/71 versus 6/77*), and mortality (1/71 versus 5/77*). We conclude that blood cardioplegia, both experimentally and clinically, offers better myocardial protection and operating conditions than does intermittent ischemia or continuous coronary perfusion.