Serum myoglobin does not predict cardiac allograft rejection.

Noninvasive studies are useful, but limited, in detecting rejection among cardiac allograft recipients. Because an elevated serum myoglobin level is a sensitive indicator of necrosis in acute myocardial infarction, we postulated that myoglobin levels might correlate with the presence, absence, or degree of rejection. Therefore we prospectively measured serum myoglobin levels at the time of endomyocardial biopsy in 45 heart transplant recipients and correlated these levels with biopsy scores (grade 0 through grade 4). There was no significant difference in mean myoglobin levels among patients with grade 0 biopsy scores and those with grade 1 through grade 4 scores. Serial myoglobin levels and endomyocardial biopsy specimens were obtained in five patients during a 4- to 9-week period; no significant directional change in myoglobin levels appeared to correlate with changes in endomyocardial biopsy score. In addition, a normal myoglobin level did not exclude, nor did an elevated level confirm, any grade of rejection. We conclude that neither the absolute level nor a directional change in serum myoglobin is useful in identifying rejection among heart transplant recipients.

Properties of pyruvate kinase and flagellar ATPase in rabbit spermatozoa: relation to metabolic strategy of the sperm cell.

Rabbit sperm pyruvate kinase remains bound to the cell structure of hypotonically treated mature rabbit epididymal spermatozoa (HTRES). It displays kinetic behavior very similar to that of rabbit muscle pyruvate kinase with regard to KM values for substrates, activation by monovalent and divalent cations, inhibition by phenylalanine which is reversed by alanine, and lack of activation by fructose-1,6-biphosphate. The flagellar ATPase also remains bound to the cell structure of HTRES, whose motility may be reactivated by a source of ATP. It requires Mg+2 for activity; the KM for both ATP and MG+2 is 0.2 mM, implying that MgATP is the substrate. The ATPase activity is not inhibited by ouabain, oligomycin, or vanadate, which also do not affect reconstituted motility, and is not affected by cyclic AMP in the presence of an inhibitor of phosphodiesterase. The activities of pyruvate kinase and the flagellar ATPase in a given preparation of HTRES are comparable. Rabbit spermatozoa have a metabolic strategy which is very similar to muscle cells. This suggests that the major use of the sperm cell's metabolic machinery is maintenance of energy for the contractile work of motility and that only minor amounts of metabolic energy appear to be consumed in other reactions, including those involved in fertilization.

Atomic absorption spectrophotometry of chromium in serum and urine with a modified Perkin-Elmer 603 atomic absorption spectrophotometer.

Modification of a Perkin-Elmer 603 atomic absorption spectrophotometer by adding a high-intensity tungsten-halogen lamp for background correction significantly improved the detection limit for elements that have analytical wavelengths in the near-ultraviolet and visible regions. Chromium in human serum and urine can be measured, with a simplified sample-handling technique, in concentrations of less than 0.1 microgram/liter. For comparison, the mean value for chromium in the serum of eight men was 0.14 microgram/liter.

Studies on cobalt myoglobins and hemoglobins. Interaction of sperm whale myoglobin and Glycera hemoglobin with molecular oxygen.

The pH dependence of the electron paramagnetic resonance (EPR) spectrum and oxygen affinity of cobaltous porphyrin-containing myoglobin (CoMb) have been examined. The hyperfine structures of the EPR spectrum of oxy-CoMb undergo small, reversible pH-dependent changes with pK values of 5.33, 5.55, and 5.25 +/- 0.05 for proto-, meso-, and deutero-CoMb's, respectively, whereas deoxy-CoMb does not exhibit any pH dependence of its EPR spectrum. The partial pressure of oxygen at half-saturation of proto-CoMb decreases from 26 to 42 Torr on lowering the pH from 7.0 to 4.8. For comparison, we have prepared cobaltous porphyrin-containing monomeric Glycera hemoglobin (CoHb (Glycera)), in which the distal histidyl group of myoglobin is replaced by a leucyl residue, and examined the equilibria and kinetics of its oxygenation and EPR spectrum. CoHb (Glycera) has exhibited a very low oxygen affinity (p50 = 7 X 10(2) Torr at 5 degrees) and a large dissociation rate constant (more than 8 X 10(4) S-1 at 5 degrees). The EPR spectrum of oxy-CoHb (Glycera) was affected by neither pH nor replacement of H2O with D2O. Low temperature photodissociation studies by EPR and spectrophotometry have shown that the photolyzed form of the ligated hemoglobin (Glycera) is similar to its deoxy form, in contrast to myoglobin which gives a new intermediate states as the photolyzed form. These differences between CoMb and CoHb (Glycera) are interpreted with relation to the possible role of the distal histidyl residue in CoMb.

Studies on cobalt myoglobins and hemoglobins. Preparation of isolated chains containing cobaltous protoporphyrin IX and characterization of their equilibrium and kinetic properties of oxygenation and EPR spectra.

Human hemoglobin containing cobalt protoporphyrin IX or cobalt hemoglobin has been separated into two functionally active alpha and beta subunits using a new method of subunit separation, in which the -SH groups of the isolated subunits were successfully regenerated by treatment with dithiothreitol in the presence of catalase. Oxygen equilibria of the isolated subunit chains were examined over a wide range of temperature using Imai's polarographic method (Imai, K., Morimoto, H., Kotani, M., Watari, H., and Kuroda, M. (1970) Biochim. Biophys. Acta 200, 189-196). Kinetic properties of their reversible oxygenation were investigated by the temperature jump relaxation method at 16 degrees. Electron paramagnetic resonance characteristics of the molecules in both deoxy and oxy states were studies at 77K. The oxygen affinity of the individual regenerated chains was higher than that of the tetrameric cobalt hemoglobin and was independent of pH. The enthalpy changes of the oxygenation have been determined as -13.8 kcal/mol and -16.8 kcal/mol for the alpha and beta chains, respectively. The rates of oxygenation were similar to those reported for iron hemoglobin chains, whereas those of deoxygenation were about 10(2) times larger. The effects of metal substitution on oxygenation properties of the isolated chains were correlated with the results obtained previously on cobalt hemoglobin and cobalt myoglobin. The EPR spectrum of the oxy alpha chain showed a distinctly narrowed hyperfine structure in comparison with that of the oxy beta chain, indicating that the environment around the paramagnetic center (the bound oxygen) is different between these chains. In the deoxy form, EPR spectra of alpha and beta chains were indistinguishable. These observations suggest that one of the inequivalences between alpha and beta chains might exist near the distal histidine group.

Energy metabolism of spermatozoa. V. The Embden-Myerhof pathway of glycolysis: activities of pathway enzymes in hypotonically treated rabbit epididymal spermatozoa.

Seven enzymes of the Embden-Myerhof pathway of glycolysis were assayed in hypotonically treated epididymal sperm from mature rabbits. These were: fructose-biphosphate aldolase, triosephosphate isomerase, glyceraldehydephosphate dehydrogenase, 3-phosphoglyceromutase, enolase, pyruvate kinase, and lactate dehydrogenase. These enzymes were firmly enough bound to the cell structure to resist removal by washing after hypotonic treatment and had maximal activities comparable to, or greater than, the rate of mitochondrial pyruvate oxidation, so that rapid oxygen uptake was observed with intermediates of the glycolytic pathway. The activity of lactate dehydrogenase in a typical preparation of hypotonically treated cells was 5.3 mumoles/minute x 10(9) cells at 25 degrees C for pyruvate reduction in the hypotonically treated cells and 4.8 mumoles/minute x 10(9) cells in the thrice-washed hypotonically treated cells. The Km for pyruvate was 1.4 mM while that for lactate was 4.4 mM. By contrast, the maximal activity of pyruvate oxidation by mitochondria was 0.10 microgram atom of oxygen/minute x 10(9) cells, corresponding to 0.020 mumole of pyruvate/minute x 10(9) cells, and the Km for pyruvate was 5 microM. These enzyme parameters favor high lactate production from glucose in aerobic glycolysis.