Red Cell Metabolism Studies on Skylab

THE UNTOWARD EFFECTS of high oxygen tension for many years had been largely of academic and in vitro interest only. Development of the use of oxygen under high pressure for medical purposes, and the use of a hyperoxic environment in the cabins of space vehicles for United States manned space flights, increased the practical implications of the potential untoward effects. These situations also provided a special opportunity for study of varying aspects of red cell metabolism. It had been demonstrated that susceptible animals exposed to oxygen under high pressure developed hemolysis due to peroxidation of unsaturated fatty acids in red cell membrane (ref. 1). It was also demonstrated that a similar event could occur in humans (ref. 2). Subsequent studies under simulated and actual space flight conditions demonstrated variable decreases of the red cell mass.

A major limiting factor in the interpretation of data obtained during the Gemini and Apollo series, however, was that blood samples were analyzed before and after flight. There was no information as to what, if any, changes occurred during space flight itself. The Skylab program thus offered a unique opportunity for the study of the possible effects of that environment and flight on red cell metabolism.

The studies carried out included an analysis of  red cell components involved with

Peroxidation of red cell lipids;

Enzymes of red cell metabolism;

Levels of 2,3-diphosphoglyceric acid and adenosine triphosphate.

Materials and Methods

The details and schedules of sampling appear elsewhere.

Blood was kept frozen for transport of samples from the Johnson Space Center to the investigator’s laboratory. Samples there remained frozen at –39 ° C (-70 ° F) until the time of determination. In all procedures, samples of blood drawn concomitantly from controls were run simultaneously with astronaut specimens.

Details of most analytic procedures used have been previously described (refs. 3,4,5,6,7,8).

The procedures employed for 2,3-diphosphoglyceric acid were basically those described by Oski (ref. 9) and Krimsky (ref. 10).

A hemoglobin determination as made on blood samples using the cyanmethemoglobin method. The final readings were thus expressed as micromoles of red cell 2,3-diphosphoglyceric acid per gram of hemoglobin. Simultaneous standards were performed with runs, and also checked against a prepared standard curve. The range used on the standard curve was between 0.1 and 0.4 micromoles (microM).

Abbreviations used in tables 27-I through 27-VI

Results

The data obtained from Skylab 2 are shown in table 27-I. Preflight differences between astronauts and controls were not noted.

With the in-flight samples, there were increases of hexokinase, pyruvate kinase, and glyceralde-hyde phosphate dehydrogenase. The changes of adenosine triphosphate and 2,3-diphosphoglyceric acid were not significant.

Postflight there was a significant decrease of phosphofructokinase.

The data obtained from Skylab 3 are summarized in tables 27-II and 27-III. Astronauts and controls were identical preflight.

During flight there were significant decreases of hexokinase, phosphoglyceric kinase, and acetyl-cholinesterase, and increases of pyruvate kinase.

Postflight the changes noted on recovery day and days 1 and 14 varied.

The results of Skylab 4 studies are shown in tables 27-IV, table 27-V and table 27-VI.

They show that the only significant change occurred in phosphofructokinase during the early stage of the in-flight samples.

Discussion

The advent of the use of medical hyperoxia and the use of increased oxygen tensions in space capsules prompted the need for further study into changes induced by variable environments and the potential untoward effects on many tissues including red cells. Previous studies in our laboratory had indicated that a mechanism, in fact the only mechanism, responsible for destruction of red cells by hyperoxia was peroxidation of the unsaturated fatty acids in red cell membranes.

In addition to these changes, other studies also demonstrated alterations of glycolytic intermediates and enzymes which, however, could not be linked to concrete evidence of cell damage.

Previous space flight studies were limited by the fact that samples could only be obtained before and after flight, and frequently inappropriate controls existed. The major contribution allowed by the Skylab series of studies was the availability of simultaneous control samples as well as in-flight samples from astronauts. It should be noted, however, that there were progressive gas composition changes as the varied series of Gemini, Apollo, and Skylab flights occurred.

In our present studies, there was no evidence of lipid peroxidation in any of the samples. This may be taken as evidence that the likelihood of overt red cell damage would be slim. There were, how-ever, certain changes observed in glycolytic intermediates and enzymes. For perspective, these are summarized in table 27-VII. Included in this table are summary data from the Skylab Medical Experiments Altitude Test (SMEAT) and our own laboratory (OHP) studies using oxygen under pressure. It is apparent that the most consistent change noted, a decrease of phosphofructokinase, had been verified a number of times. It is this enzyme step which is thought to be at the center of the so-called Pasteur effect, and which is susceptible to the effects of oxygen. Other changes have been less consistent and the significance of all of these changes is not understood.

Summary

In summary therefore, it is possible to conclude that there are no evidences of lipid peroxidation, that biochemical effect known to be associated with irreversible red cell damage, and the changes observed in glycolytic intermediates and enzymes cannot be directly implicated as indicating evidence of red cell damage.

References

1. MENGEL, C.E., and H.E. KANN, JR. Effects in vivo hyperoxia on erythrocytes. III. In vivo peroxidation of erythrocyte lipid. J. Clin. Invest., 45:1150-1158, 1966.

2. MENGEL, C.E., H.E. KANN, JR., A. HEYMAN, and E. METZ. Effects of in vivo hyperoxia on erythrocytes. II. Hemolysis in a human after exposure to oxygen under high pressure. Blood, 25:822-829, 1966.

3. ZIRKLE, L.G., Jr., C.E. MENGEL, S.A. BUTLER, and R. FUSON. Effects of in vivo hyperoxia on erythrocytes. IV. Studies in dogs exposed to hyperbaric oxygenation. Proc. Soc. Exp. Biol. & Med., 119:833-837, 1965.

4. O’MALLEY, B.W., C.E. MENGEL, W.D. MERIWETHER, and L.G. ZIRKLE, JR. Inhibition of erythrocyte acetylcholinesterase by peroxides. Biochemistry, 5:40-44,1966.

5. KANN, H.E., JR., and C.E. MENGEL. Mechanisms of erythrocyte damage during in vivo hyperoxia. Proc. 3^rd Int. Conf. on Hyperbaric Medicine, pp. 65-72. November, 1966.

6. O’MALLEY, B.W., and C.E. MENGEL. Effects of in vivo hyperoxia on erythrocytes. V. Changes of RBC glycolytic intermediates in mice after in vivo oxygen under high pressure. Blood, 29:196-202, 1967.

7. TIMMS, R., and C.E. MENGEL. Effects of in vivo hyperoxia on erythrocytes. VII. Inhibition of RBC phosphofructokinase. Aerospace Med., 39:71-73,1968.

8. SMITH, D., R. TIMMS, C.E. MENGEL, and D. JEFFERSON. Effects of in vivo hyperoxia on erythrocytes. VIII. Effect of adenosine triphosphate (ATP) and related glycolytic enzymes. Johns Hopkins Med. J., 122:168-171, 1968.

9. OSKI, F.A., A.J. GOTTLIEB, D. MILLER, and M. DELIVORIA-PAPADOPOULOS. The effects of deoxyhemoglobin of adult and fetal hemoglobin on the synthesis of red cell 2,3-diphosphoglyceric acid and its in vivo consequences. J.Clin. Invest., 48:400, 1970.

10. KRIMSKY, I. 2,3-diphosphoglycerate. In Enzymatic Analysis. Bergmeyer, H.U. (Ed.) Academic Press, Inc. New York, 1963. (1^st edition.)

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