Hypotensive resuscitation using a polymerized bovine hemoglobin-based oxygen-carrying solution (HBOC-201) leads to reversal of anaerobic metabolism.

BACKGROUND: Traditional resuscitation regimens have been recently challenged. This study evaluates hypotensive resuscitation with a hemoglobin-based oxygen-carrying (HBOC) solution after severe hemorrhage in a porcine model. We hypothesized that HBOC-201 restores tissue perfusion at a lower mean arterial pressure than standard resuscitation fluids. METHODS: Yorkshire swine (55-65 kg, n = 30), were rapidly hemorrhaged to a mean arterial pressure (MAP) of 30 mm Hg, maintained hypotensive for 45 minutes, and randomized into groups. Group I was resuscitated with an HBOC solution to a MAP of 60 mm Hg. Groups II and III were resuscitated to a MAP of 80 mm Hg with lactated Ringer's solution (LR) alone or LR (40 mL/kg) followed by shed blood, respectively. Group IV was resuscitated with shed blood alone to a MAP of 60 mm Hg. Group V received an HBOC solution to a MAP of 50 mm Hg. Hemodynamic variables, Swan-Ganz parameters, blood gas samples, and lactate levels were followed for 5 hours. Data were analyzed by analysis of variance/Duncan multiple range test. RESULTS: There were no significant differences in mortality between any groups. Groups I, IV, and V had lower (p < 0.05) cardiac output, pulmonary artery wedge pressure, and MAP than either group II or group III. Svo2 was significantly lower in the HBOC groups. There were no significant differences in arterial pH or lactate between groups I, III, and IV. Lactate levels, base excess, and arterial pH were significantly worse in the LR-alone and HBOC-50 groups. CONCLUSION: Hypotensive resuscitation with HBOC-201 at a MAP of 60 mm Hg after a controlled hemorrhage in swine provides sufficient tissue perfusion and oxygen delivery to reverse anaerobic metabolism on the basis of global physiologic markers despite continued hypotension, hypovolemia, and low cardiac output.

Calpromotin, a cytoplasmic protein, is associated with the formation of dense cells in sickle cell anemia.

We have tested the hypothesis that dense cell formation in sickle cell disease is associated with increased binding of calpromotin to the membrane, an event that occurs during the activation of calcium-dependent potassium transport. By SDS polyacrylamide gel electrophoresis, we found that sickle cell membranes contained more calpromotin than did normal membranes when stained with Coomassie brilliant blue or when transferred to nitrocellulose paper and immunostained with horseradish peroxidase. Also, the membranes from dense sickle cells contained significantly (P = 0.00055) higher levels of calpromotin, 2.62+/-1.59 microg/mg membrane protein, compared to light sickle cells, 1.40+/-0.70 microg/mg membrane protein, when measured by an enzyme-linked immunosorbent assay. The ratio of calpromotin associated with dense cell membranes to light cell membranes was significantly greater than 1.0 (P < 0.00005). Transmission electron micrographs of immunogold-labelled membranes supported the increase in calpromotin binding in dense sickle cell membranes. In addition, the immunogold probe demonstrated clustering, which was not observed in light sickle cell membranes nor in normal membranes. Finally, we incubated HbSS cells in vitro using a repetitive deoxygenation/ reoxygenation procedure to produce dense cells and then measured the levels of calpromotin associated with their membranes. As expected, the levels of calpromotin bound to the membrane doubled during the procedure relative to the basal levels at the beginning of the incubation. The correlation coefficient, calculated between the increase in dense cell formation and the increase in calpromotin associated with the membrane, was statistically significant (P = 0.038). The results demonstrate that an increase in calpromotin binding to the membrane is associated with dense cell formation presumably through the activation of the calcium-dependent potassium channel.

Do providers adhere to ACOG standards? The case of prenatal care.

OBJECTIVE: To examine the extent to which obstetric providers abide by prenatal practice guidelines published by ACOG. METHODS: The prenatal records were abstracted for low-risk patients initiating care with randomly selected urban obstetrician-gynecologists, rural obstetrician-gynecologists, urban family physicians, rural family physicians, and urban certified nurse-midwives in Washington state between September 1, 1988 and August 30, 1989. The prenatal care recorded in their medical charts was compared with the ACOG-recommended guidelines. RESULTS: Overall, providers of all five types adhered closely to the published standards. Certified nurse-midwives recorded a standard of practice that most closely matched that recommended by ACOG. Overall, there was less complete adherence in the recording of maternal height, fetal activity after 30 weeks' gestation, and fetal presentation at or after 36 weeks' gestation. Those laboratory tests that ACOG has recommended most recently (serum alpha-fetoprotein and diabetes screening) and those not recommended for routine use were ordered less often on average by providers. CONCLUSIONS: The cross-sectional nature of this study cannot demonstrate definitively that ACOG's guidelines have changed provider prenatal practices. However, these findings demonstrate that providers in varying specialties and geographic locations can adhere to a detailed set of clinical guidelines if they are appropriately disseminated and implemented.

Increased susceptibility of the sickle cell membrane Ca2+ + Mg(2+)-ATPase to t-butylhydroperoxide: protective effects of ascorbate and desferal.

Normal and sickle cell erythrocyte membranes were examined for significant differences in their ATPase activities, thiobarbituric acid reactive products formed (measured relative to malondialdehyde), membrane protein polymerization, and number of protein-free sulfhydryl groups when treated with 0.5 mmol/L t-butylhydroperoxide (tBHP) for 30 minutes. Isolated sickle cell membranes treated with tBHP produced significantly greater inhibition in both their basal and calmodulin-stimulated Ca2+ + Mg(2+)-ATPase activities (75% inhibition in both cases) compared with that of control membranes. In addition, there was significantly more malondialdehyde formed from sickle cell membranes compared with control membranes. Oxidation caused greater protein polymerization in sickle cell membranes compared with normal membranes as demonstrated by the formation of high molecular weight polymers separated on sodium dodecyl sulfate polyacrylamide gels. The number of free sulfhydryl groups present in spectrin and actin decreased more in sickle cell membranes as measured by 3H-N-ethyl maleimide autoradiography and gel scanning. To prevent enzyme inhibition, erythrocyte membranes were treated with tBHP in the presence of 1 mmol/L ascorbate, a potential antioxidant, and 1 mmol/L desferal, an iron chelator. Both ascorbate and desferal added alone with tBHP were effective in preventing inhibition of the basal and calmodulin-stimulated Ca2+ + Mg(2+)-ATPase activities in normal membranes, but in sickle cell membranes only the addition of ascorbate and desferal together offered significant protection. The enhanced oxidation observed with sickle cell membranes can be mimicked in normal white membranes by adding hemoglobin, hemin, or ferrous chloride in the presence of tBHP. In contrast to hemoglobin, ferrous chloride has the ability to enhance membrane oxidation in the presence of ascorbate with or without tBHP. Furthermore, the addition of desferal to these membranes greatly decreased the iron-ascorbate-tBHP oxidation of erythrocyte membranes as determined by the sustained ATPase activities and the reduced formation of malondialdehyde. Maximal protection was provided by 1 mmol/L desferal in the presence of 1 mmol/L ascorbate, although some protection was observed even at 10 mumol/L, the lowest concentration tested. These results are discussed in light of the pro- and anti-oxidant effects of ascorbate in the absence and presence of iron and tBHP.

Ascorbate protects against tert-butyl hydroperoxide inhibition of erythrocyte membrane Ca2+ + Mg2(+)-ATPase.

The incubation of erythrocyte suspensions or isolated membranes containing a residual amount of hemoglobin (0.04% of original cellular hemoglobin) with tert-butyl hydroperoxide (tBHP, 0.5 mM) caused significant inhibition of basal and calmodulin-stimulated Ca2+ + Mg2(+)-ATPase activities and the formation of thiobarbituric acid reactive products measured as malondialdehyde. In contrast, the treatment of white ghosts (membranes not containing hemoglobin) with tBHP (0.5 mM) did not lead to appreciable enzyme inhibition within the first 20 min and did not result in malondialdehyde (MDA) formation. However, the addition of either 10 microM hemin or 100 microM ferrous chloride + 1 mM ADP to white ghosts produced hydroperoxide effects similar to those in pink ghosts (membranes with 0.04% hemoglobin). The concentrations of hemin and ferrous chloride which caused half-maximal inhibition of Ca2+ + Mg2(+)-ATPase activity at 10 min were 0.5 and 30 microM, respectively. The effects of several antioxidants (mannitol, thiourea, hydroxyurea, butylated hydroxytoluene, and ascorbate) were investigated for their protective effects against oxidative changes resulting from tBHP treatment. Over a 30-min incubation period only ascorbate significantly reduced the enzyme inhibition, MDA formation, and protein polymerization. Thiourea and hydroxyurea decreased MDA formation and protein polymerization but failed to protect against the enzyme inhibition. Butylated hydroxytoluene was similar to thiourea and hydroxyurea but with better protection at 10 min. Mannitol, under these conditions, was an ineffective antioxidant for all parameters tested.

Inhibition of T-lymphocyte activation by amiloride.

The T-lymphocyte activation process involves a series of coordinately coupled biochemical events occurring in response to antigen or mitogen. These events have not been completely characterized. The present studies investigate the mechanism of protein synthesis during the initial phase of T-cell activation. Among the early biochemical changes, induction of protein synthesis was observed as early as 10 minutes after mitogen stimulation of T-lymphocytes. This early protein synthesis was inhibited by cycloheximide but was insensitive to actinomycin-D, indicating the presence of preformed mRNA in resting lymphocytes. Since early protein synthesis parallels the increase in protein kinase C activity in activated T-lymphocytes, these two biochemical events may be related. In the present report, amiloride, an inhibitor of Na+/H+ antiport and protein kinase C, significantly inhibited [3H]leucine and [3H]thymidine incorporation in a dose-dependent manner into phytohemagglutinin (PHA)-stimulated T-lymphocytes. Furthermore, when T-lymphocytes were stimulated by phorbol myristate acetate, a known activator of protein kinase C, a similar inhibition of protein and DNA synthesis by amiloride was observed. The partially purified cytosol fraction isolated from PHA-activated T-lymphocytes showed a 75% decrease in protein kinase C-mediated [32P] incorporation from ATP in the presence of 100 microM amiloride. These results suggest that the T-cell activation process following exposure to mitogens involves early protein synthesis, which may be mediated by protein kinase C.

Effect of polymyxin-B on T-lymphocyte protein synthesis.

The role of protein kinase-C (PK-C) protein phosphorylation on the mitogen triggered responses of T-lymphocytes was examined by observing the effect of polymyxin-B (an inhibitor of PK-C) on mitogen induced protein and DNA synthesis. Polymyxin-B inhibited 3H-thymidine incorporation by PHA activated T-lymphocytes over a range of PHA concentrations. 3H-leucine incorporation by PHA activated T-lymphocytes was inhibited by polymyxin-B in a dose dependent manner. A partially purified PK-C fraction from polymyxin-B treated PHA activated T-lymphocytes demonstrated less than 25% of the phosphorylating activity of untreated lymphocytes. These results suggest that protein synthesis during the T-lymphocyte activation process is dependent on PK-C activity.

Depressed erythrocyte glutathione reductase activity in sickle cell disease.

In a group of 27 sickle cell disease patients ranging in age from 2 yr 3 months to 43 yr, 13 (48%) were found to have depressed erythrocyte glutathione reductase activity suggesting riboflavin deficiency. Glutathione reductase activity coefficients did not correlate with riboflavin intakes which were calculated from 3-day diet records returned by 16 patients. Other causes of riboflavin deficiency including decreased absorption, altered metabolism, or increased excretion of the vitamin must be considered. The potential effect of depressed erythrocyte glutathione reductase activity in the sickle cell disease process is discussed.

The weight of the mature wheat grain.

Grains retained on the plants of some cultivars of common bread wheat lose dry matter after ripeness is attained, but later gain dry matter again. We conclude that the post-ripe plant may remain metabolically active.