A comparison of the GroE chaperonin requirements for sequentially and structurally homologous malate dehydrogenases: the importance of folding kinetics and solution environment.

Escherichia coli malate dehydrogenase (EcMDH) and its eukaryotic counterpart, porcine mitochondrial malate dehydrogenase (PmMDH), are highly homologous proteins with significant sequence identity (60%) and virtually identical native structural folds. Despite this homology, EcMDH folds rapidly and efficiently in vitro and does not seem to interact with GroE chaperonins at physiological temperatures (37 degrees C), whereas PmMDH folds much slower than EcMDH and requires these chaperonins to fold to the native state at 37 degrees C. Double jump experiments indicate that the slow folding behavior of PmMDH is not limited by proline isomerization. Although the folding enhancer glycerol (<5 m) does not alter the renaturation kinetics of EcMDH, it dramatically accelerates the spontaneous renaturation of PmMDH at all temperatures tested. Kinetic analysis of PmMDH renaturation with increasing glycerol concentrations suggests that this osmolyte increases the on-pathway kinetics of the monomer folding to assembly-competent forms. Other osmolytes such as trimethylamine N-oxide, sucrose, and betaine also reactivate PmMDH at nonpermissive temperatures (37 degrees C). Glycerol jump experiments with preformed GroEL.PmMDH complexes indicate that the shift between stringent (requires ATP and GroES) and relaxed (only requires ATP) complex conformations is rapid (<3-5 s). The similarity in irreversible misfolding kinetics of PmMDH measured with glycerol or the activated chaperonin complex (GroEL.GroES.ATP) suggests that these folding aids may influence the same step in the PmMDH folding reaction. Moreover, the interactions between glycerol-induced PmMDH folding intermediates and GroEL.GroES.ATP are diminished. Our results support the notion that the protein folding kinetics of sequentially and structurally homologous proteins, rather than the structural fold, dictates the GroE chaperonin requirement.

Immunodetection of hepatic peroxisomal PMP70 as an indicator of peroxisomal proliferation in the mummichog, Fundulus heteroclitus.

Peroxisomes are important sites for beta-oxidative fatty acid metabolism and peroxidative detoxification. Agents causing peroxisomal proliferation have been associated with reproductive and developmental toxicity and hepatocarcinogenesis. Female mummichog (Fundulus heteroclitus) were exposed to waterborne 2,4-dichlorophenoxyacetic acid (2,4-D), a model peroxisome proliferator, at sublethal concentrations of 0.01, 0.10, and 1.00 ppm or dimethyl sulfoxide (DMSO) vehicle for 7, 14, or 21 days. A polyclonal antibody to rat PMP70 protein (70 kDa peroxisomal membrane protein, a major component of peroxisomes and member of the ABC transporter superfamily) was used for Western blotting after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to determine possible peroxisome proliferation. Significant increases of an approximately 70-kDa protein band recognized by anti-PMP70 were observed on all days, especially at the highest exposure concentration. The data suggest that immunoassay of PMP70 is a useful biomarker assay for peroxisome proliferation, and may be applicable to a wide range of species. The response also suggests that this assay could be used for measuring chronic exposures to environmental peroxisome proliferating agents.

Blood transfusion-acquired hemoglobin C.

Unexpected and confusing laboratory test results can occur if a blood sample is inadvertently collected following a blood transfusion. A potential for transfusion-acquired hemoglobinopathy exists because heterozygous individuals show no significant abnormalities during the blood donor screening process. Such spurious results are infrequently reported in the medical literature. We report a case of hemoglobin C passively transferred during a red blood cell transfusion. The proper interpretation in our case was assisted by calculations comparing expected hemoglobin C concentration with the measured value. A review of the literature on transfusion-related preanalytic errors is provided.

Efficacy of intraoperative blood salvage during coronary artery bypass grafting.

OBJECTIVE: To analyze the usefulness of autologous blood salvage and intraoperative isovolemic hemodilution in conjunction with autologous blood salvage in cardiac surgery, and also to compare two different autologous blood salvage methods. EXPERIMENTAL DESIGN: Retrospective analysis of transfusion practice in cardiac surgery. SETTING: University hospital. PATIENTS: One hundred and sixty two consecutive patients who had coronary artery bypass grafting (CABG) from January to September, 1993. INTERVENTIONS: A review of transfusion records of patients who underwent intraoperative autologous blood salvage and isovolemic hemodilution. MEASURES: Perioperative transfusion requirements were analyzed in patients undergoing CABG using a cell saver system whereby blood was collected, processed and reinfused to the patients in the operating room. RESULTS: Nineteen percent of patients did not receive perioperative homologous blood transfusion when a cell saver was used intraoperatively. When autologous isovolemic hemodilution was used in conjunction with a cell saver, fifty-four percent of the patients did not receive homologous blood transfusion. The results of this study were compared to matched patients undergoing CABG using a different type of cell saver system whereby blood was collected in the operating room, processed in the blood bank and reinfused in the intra- and/or postoperative period. We found that the former system provided a more timely and greater yield of blood salvaged and as a consequence, less homologous blood transfusion. CONCLUSIONS: Intraoperative blood salvaging provides an immediate source of red blood cells and decreases the utilization of homologous blood. In addition, isovolemic hemodilution in conjunction with blood salvage further decreases homologous blood transfusion needs.

Improving the efficacy of preoperative autologous blood donation in patients with low hematocrit: a randomized, double-blind, controlled trial of recombinant human erythropoietin.

The effects of therapy with recombinant human erythropoietin (Epoetin alfa) on erythropoiesis, preoperative autologous blood donation, and risk of exposure to allogeneic blood were evaluated in 204 patients scheduled to undergo elective orthopedic surgery. Study protocol required patients to have a baseline hematocrit < or = 39% and surgery scheduled 25-35 days in advance. Patients were randomized to two equal groups and were seen at study centers every 3-4 days within the 21-day trial period. At each visit, phlebotomy(< or = 450 mL) was performed if the hematocrit was > or = 33%, and Epoetin alfa (600 U/kg) or placebo was administered intravenously. A total of 173 patients were assessable; 31% of placebo recipients and 20% of Epoetin alfa recipients required allogeneic transfusion (p = 0.09). Logistic regression modeling showed that the risk of allogeneic transfusion was reduced by Epoetin alfa (p = 0.025). When patients receiving > 6 units of blood (necessitating allogeneic units) were excluded from analysis, 29% of placebo recipients and 14% of Epoetin alfa recipients were exposed to allogeneic blood (p = 0.015). Epoetin alfa recipients predonated more autologous units than did placebo recipients (4.5 vs 3.0 units, respectively; p < 0.001), and their production of red blood cells increased significantly more over baseline production values (668 vs 353 mL, respectively; p < 0.05). These results demonstrate that administration of Epoetin alfa stimulates erythropoiesis, allows predonation of more units of autologous blood, and reduces the risk of exposure to allogeneic blood. Optimal dosing regimens and surgical patients most likely to benefit fro Epoetin alfa therapy must be established.

The effect of recombinant human erythropoietin on the efficacy of autologous blood donation in patients with low hematocrits: a multicenter, randomized, double-blind, controlled trial.

BACKGROUND: This randomized controlled study was undertaken to determine the effect of recombinant human erythropoietin (rHuEPO) on erythropoiesis, autologous blood collection, and allogeneic transfusion risk in elective surgery patients with low baseline hematocrits. STUDY DESIGN AND METHODS: Patients (n = 204) with low baseline hematocrits ( < or = 39%), scheduled for orthopedic surgery within 25 to 35 days, were seen every 3 to 4 days for 21 days. At each visit, 450 mL of blood was collected if the hematocrit was > or = 33 percent, and rHuEPO (600 U/kg) or placebo was administered intravenously. RESULTS: One hundred seventy-three patients were evaluable. The number of autologous units collected from the rHuEPO and control groups, respectively, was 4.5 +/- 1.0 and 3.0 +/- 1.1 (p < 0.001), and marrow production of red cells increased by 668 +/- 222 and 353 +/- 155 mL over and above baseline production (p < 0.05). Allogeneic blood transfusion was required by 31 percent of control and 20 percent of rHuEPO patients (p = 0.09). Excluding 8 patients who received > 6 units, 29 percent of control and 14 percent of rHuEPO patients required allogeneic blood (p = 0.015). Logistic regression modeling determined that the risk of allogeneic transfusion was reduced by rHuEPO (p = 0.025). CONCLUSION: The use of rHuEPO stimulates erythropoiesis, permits the storage of more autologous blood, and reduces allogeneic transfusion risk in patients with low hematocrits who are undergoing elective orthopedic surgery. Additional studies are necessary to determine the optimal schedules of rHuEPO administration and autologous blood collection as well as the cost-effectiveness of this strategy.

Comparing the transfusion medicine content of the NBME's examinations in 1984-1985 and 1989-1990.

Recognition of the seriousness of transfusion-transmitted diseases has been demonstrated by U.S. medical schools through the integration of transfusion medicine (TM) content into their curricula. To evaluate the degree to which these changes in curricula have been reflected in the National Board of Medical Examiners' (NBME) examinations, a study conducted in 1991 evaluated the proportions of TM-related items on Parts I and II of the NBME examinations for 1984-1985 versus 1989-1990. Both Part I (basic sciences) and Part II (clinical sciences) demonstrated significant gains in TM items between the comparison periods (p less than .001), with Part II having the higher gain. An analysis of students' knowledge revealed that students in 1989-1990 tended to perform better on TM items than on examination items generally. The increases in TM content and student performance on TM items on the 1989-1990 examinations suggest that the national effort to expand and improve teaching of TM in U.S. medical schools has been effective.

Intraoperative autologous blood salvage with cardiac surgery: an analysis of five years' experience in more than 3,000 patients.

STUDY OBJECTIVE: To analyze intraoperative autologous salvage of shed mediastinal blood and subsequent transfusion in cardiac surgery. DESIGN: Retrospective statistical analysis. SETTING: University hospital. PATIENTS: Three thousand twenty two patients undergoing cardiac surgery from 1984 to 1988. INTERVENTIONS: A review of anesthesia and transfusion records of all patients who underwent intraoperative salvage of shed blood and autologous transfusion using the Sorenson Receptal Auto Transfusion System (ATS) with saline wash prior to reinfusion in cardiac surgery. MEASUREMENTS AND MAIN RESULTS: The salvaged blood volume ranged from 36 to 2,795 ml, with a mean of 321 +/- 222 ml (SD). Eighteen percent of patients did not receive any homologous blood products during their hospitalization. Patients who received only salvaged autologous transfusion were younger, had higher preoperative hemoglobin and hematocrit values, had a larger body surface area, and had shorter surgeries compared with patients who received only homologous blood or both autologous and homologous blood. More blood products were given to patients who received salvaged autologous blood compared with those who did not. Patients who underwent normovolemic hemodilution prior to extracorporeal circulation with subsequent reinfusion received significantly fewer blood products. Ten preoperative and four intraoperative variables significantly influenced the salvaged volume. Previous cardiac surgery was the most significant preoperative variable, and repair of ventricular septal defect produced by myocardial ischemia was the most significant intraoperative variable. CONCLUSION: Considering the average salvaged volume and its current autologous transfusion-related expense, autologous blood salvage is potentially an economic benefit. Perioperative blood conservation requires a considerable commitment from surgeons, anesthesiologists, perfusionists, and intensive care physicians to be effective.

The variability of transfusion practice in coronary artery bypass surgery. Transfusion Medicine Academic Award Group.

We audited 540 patients undergoing elective first-time coronary artery bypass grafts at 18 institutions. The purposes of the study were to describe the variability in transfusions among institutions and to determine factors that may account for variability. Mean homologous red blood cell use per patient was 2.9(+/- 0.1) U (institutional range, 0.4 to 6.3 U). One hundred seventy-seven patients (32%) received plasma (institutional range, 0% to 97%), and 119 (22%) received platelets (institutional range, 0% to 80%). After controlling for patient and surgical practice variables, transfusion practice factors still accounted for variation in red blood cell transfusions. Variation in patients receiving plasma and platelet transfusions among institutions was determined in part by prophylactic transfusions. We conclude that blood component usage for coronary artery bypass grafts differs widely among institutions. The variability in use of these components is accounted for in part by unnecessary transfusions in otherwise routine, uncomplicated coronary artery bypass graft procedures.

Guidelines for transfusion support in patients undergoing coronary artery bypass grafting. Transfusion Practices Committee of the American Association of Blood Banks.

We have reviewed the impact of evolving issues in coronary artery bypass grafting (CABG) on transfusion support for these patients. Issues include increased awareness of transfusion risks, reappraisal of traditional indicators triggering transfusion, and evolving alternatives to homologous blood transfusion such as autologous blood and pharmacologic therapy. These issues have been prompted by programs, such as the National Institutes of Health Consensus Conferences, to provide physicians with guidelines for appropriate use of blood components. However, evidence suggests that transfusion practice in coronary artery bypass grafting procedures remains variable and does not take into account the results of recently published clinical studies. We have therefore developed guidelines and recommendations for transfusion support in patients undergoing coronary artery bypass grafting. In summary, they are the following. 1. Institutions with coronary artery bypass grafting programs should establish a multidisciplinary approach to use a combination of interventions designed to minimize homologous blood exposure. 2. Prophylactic transfusion of plasma and platelets are of no benefit and therefore carry an unnecessary risk to the patient. 3. Special request products such as designated blood donation from first-degree relatives should not be used because of the risk of transfusion-associated graft versus host disease. 4. For support of intravascular volume, crystalloids or colloids should be used because they do not have the potential to transmit infection.

Strategies for the review of transfusion practices.

The intent of this report is to familiarize health care professionals with the concept of effective quality assurance in regard to blood use. Although evaluation of the appropriateness of transfusion therapy is now required by the Joint Commission on Accreditation of Health Organizations, health care facilities have little experience with this aspect of professional quality assurance. To this end, the Committee on Transfusion Practices of the American Association of Blood Banks, in Arlington, Va, in this report has provided examples of indications and audit criteria for individual blood components and products and commented on areas of controversy surrounding their use. Audit criteria from different institutions may vary because of differences in local interpretation of the indication, different patient populations, and, in some instances, the availability of blood and laboratory services. Several approaches to the review of transfusion practices are discussed in relation to clinical settings and pertaining to particular blood components. It is evident from these examples that there will be an increased need for trained personnel to perform the initial review process as well as for physicians trained in transfusion medicine to oversee the transfusions and provide the necessary consultation.

Detection of cold hemagglutination in a blood cardioplegia unit before systemic cooling of a patient with unsuspected cold agglutinin disease.

A case is described in which hemagglutination occurred intraoperatively in cold blood flushed through the blood cardioplegia delivery system from a patient with unsuspected cold agglutinin disease. On initiating cardiopulmonary bypass and then selectively cooling the perfusate in the blood cardioplegia delivery system before inducing systemic cooling, it is possible to check for cold agglutination. Routine use of this technique may be worthwhile to detect cold agglutination in vitro before systemic cooling is begun in the rare patient with unsuspected cold agglutinins.

A two-institution study of transfusion practice in 78 consecutive adult elective open-heart procedures.

Blood salvage techniques and increasingly conservative physician transfusion practice in cardiac surgery have led to reports of homologous blood exposure in as few as 10% of patients having elective cardiac revascularization surgery (Ann Thorac Surg 1985;40:380). To identify current prevailing transfusion practice between centers, the authors prospectively audited 49 and 29 consecutive adult elective open-heart surgery cases (78 total) at two centers. Thirty-six of 49 patients (73%) received 245 homologous blood units (HBs) at institution 1 (m = 5.0). Fifteen of 29 (52%) received 84 HBs at institution 2 (m = 2.9, P less than 0.05). Sex, age, duration of surgery, intraoperative blood salvaged, preoperative hematocrit (Hct), nadir Hct, and nadir platelet counts, and the surgeon were all found to be determinants of transfusion practice at institution 1 but not at the other. The data indicate that (1) determinants of homologous blood exposure are not consistent between institutions and instead reflect "prevailing practice" rather than need; (2) a prospective multi-institution audit of prevailing transfusion practice in cardiac revascularization is needed to address this; (3) autologous blood pre-deposit is underused in open-heart surgery.

Determination of need for elution studies for positive direct antiglobulin tests in pretransfusion testing.

Retrospective evaluation of elution studies for specimens with positive direct antiglobulin test (DAT) results during a three-month interval revealed 29 warm autoantibodies and 28 nonreactive eluates. Clearly, routine elution studies were nonproductive. However, there was no evaluation method for exclusion from elution studies. Therefore, an algorithm correlating laboratory, transfusion, and clinical data was developed. It identified delayed hemolytic transfusion reactions and possible autoimmune hemolytic anemia. By following the algorithm, technologists would perform elution studies, forego them, or consult the Medical Director. To test the algorithm, 568 positive DATs identified from 12,416 specimens were analyzed. Technologist evaluation without medical consultation for 194 specimens yielded 106 elution studies; 374 specimens required medical consultation, and 166 had eluates. Seven delayed hemolytic transfusion reactions were found. This suggests certain elution studies can be eliminated routinely. However, DATs with positive results associated with possible delayed hemolytic transfusion reactions require investigation. The search for autoimmune hemolytic anemia was not productive.

Transfusion therapy in emergency medicine.

Volume replacement is critical to the resuscitation of the hemorrhaging patient, but this usually can be accomplished quickly and safely with crystalloid and/or colloid solutions. Red cells should be used in addition to asanguinous fluids in the treatment of tissue hypoxia due to anemia. The need for whole blood as opposed to packed red blood cells is controversial. However, plasma should not be used as a volume expander, and its use to supplement coagulation factors during the massive transfusion of red cells should be guided by laboratory tests that document a coagulopathy. Similarly, platelet transfusions are indicated to correct documented thrombocytopenia or platelet dysfunction, and routine prophylaxis after fixed volumes of red cells results is unwarranted. Many anticipated complications of massive transfusions, including hemostatic abnormalities, acid-base imbalances, hyperkalemia, and hypocalcemia, are uncommon or of limited clinical significance. The risks of immune hemolysis and transfusion-transmitted diseases, on the other hand, are significant, and argue for judicious use of blood components. In emergencies in which blood is required immediately before compatibility testing can be completed, O-negative uncrossmatched blood can be requested. Careful blood specimen collection and patient identification prior to transfusion are critical. Practices that emphasize blood conservation, including the use of autologous salvaged blood, are always to the patient's advantage.

Adrenocorticotropic hormone causes long-lasting potentiation of transmitter release from frog motor nerve terminals.

Exposure of frog neuromuscular preparations to adrenocorticotropic hormone for several minutes increased both nerve-evoked and spontaneous transmitter release for several hours. No changes in postsynaptic sensitivity to transmitter were detected. The long-lasting potentiation shows little sensitivity to changes in extracellular calcium concentration and seems to be entirely presynaptic in origin.

Electrotonic coupling in internally perfused crayfish segmented axons.

We have developed a technique for cannulation and internal perfusion of crayfish segmented lateral axons. Experiments on perfused and non-perfused axons lead to the following conclusions: 1. Internally perfused segmented axons behave very similarly to non-perfused axons. 2. The axial electrical resistance of the junctional region is almost as low as a comparable segment of axon. 3. Neither intracellular Ca2+ nor H+ is effective in disrupting the intercellular communication pathway in perfused axons. On the basis of these findings we have formulated a hypothesis for cellular control of intercellular coupling based on the existence of a soluble intermediate for Ca2+ or H+-induced uncoupling. This hypothesis is consistent with data from both internally perfused and non-perfused axons.

Interaction of anaesthetics with electrical synapses.

Studies of the interaction of anaesthetics with various preparations, from whole animals to organic solvents, have been continuing since Overton and Meyer found a correlation between anaesthetic potency and solubility in olive oil. Although the physiological basis of anaesthesia is far from clear, one popular hypothesis is that anaesthetics act primarily by interfering with the normal functioning of chemical synapses. This hypothesis is supported by experiments showing that these synapses are more sensitive to both local and general anaesthetics than are axons. The effects of anaesthetics on electrical synapses (gap-junctions or nexus) have not previously been studied. These ubiquitous structures, presumably responsible for cell-to-cell communication, are found in most vertebrate and invertebrate tissues. We report here the effects of several anaesthetics on electronic coupling between nerve cells, and show that electrical synapses are less sensitive to most anaesthetics than are chemical synapses and axonal membranes.