Fred J. Oelshlegel

Effect of zinc on increasing oxygen affinity of sickle and normal red blood cells

Abstract We have hypothesized a state of zinc deficiency in sickle cell disease (SCD). This could at least partially explain the growth problems, hypogonadism, and slow healing leg ulcers associated with SCD. Preliminary findings revealed abnormally low red blood cell zinc levels in 10 of 16 patients studied. Before suggesting zinc supplementation in SCD we thought it important to look at the effect of zinc on red cell metabolism and function. It was found that zinc chloride added to normal and SCD blood to a final concentration of 1.5 × 10 −3 M caused a left-shift of the blood oxygen affinity curve (increased oxygen affinity) varying from 1.5 to 3.5 mm Hg change in half saturation (p50). This curve shifting property has important implications for SCD since recent work with cyanate suggests that such shifts are very beneficial in treatment of SCD. Thus zinc supplementation in SCD, in addition to its potential role in correcting wound healing and growth problems, may have a beneficial effect on the basic pathological process. Data are given which suggest that zinc and 2, 3-diphosphoglycerate may not be competing for the same site on the hemoglobin molecule.

Studies on the interaction of zinc with human hemoglobin

Abstract Zn has previously been shown to increase the oxygen affinity of both normal and sickle red blood cells. Experiments are presented which demonstrate that the oxygen affinity effect of Zn is due to a Zn-hemoglobin binding mechanism rather than a Zn-2,3 diphosphoglycerate binding mechanism. Further a large shift (6 mm Hg) in the oxygen affinity of a red cell-saline suspension occurs with a low Zn/hemoglobin (tetramer) molar ratio (0.4). Zn had no influence on the Bohr effect of hemoglobin but it did decrease the Hill coefficient. Hemoglobin binding experiments using partially purified hemoglobin indicated that Zn can bind to more than one amino acid residue but it appears that the amino acid residue with the highest binding capacity for Zn is also the residue involved in the oxygen affinity effect of Zn. Hydrogen ion concentration (pH 5–8) had no influence on the Zn/hemoglobin ratios obtained in these binding experiments. The possible (and the improbable) Zn binding sites on the hemoglobin molecule are discussed.

Quantitative analysis of pyridine nucleotides in red blood cells: A single-step extraction procedure

Abstract Quantitative analysis of red cell pyridine nucleotides has been unreliable in the past because of technical problems in extracting them in the presence of hemoglobin. A simple alcoholic extraction procedure for analysis of pyridine nucleotides in red blood cells is described in this paper. Pyridine nucleotides extracted in the presence of hemoglobin in solution show recoveries of NADH, NAD, and NADP averaging over 70%, while recoveries of NADPH were about 60%. In order to show that these techniques could detect actual intracellular differences in nucleotides inside red cells, two experiments were performed in which the ratios of the nucleotides would be predictably altered. Intact cells incubated in the presence of methylene blue show a decrease in the NADPH NADP ratio, and intact cells incubated in the presence of hydrazine and lactate show an increase in the NADH NAD ratio. The changes in pyridine nucleotide ratios in these experiments are in the expected direction and were easily detected. Levels of pyridine nucleotides in red blood cells of normal human adults are also presented.

New positive, tetrazolium-linked, staining method for use with electrophoresis of phosphoglycerate kinase

Eine Methode zur positiven Anfärbung der Phosphoglycerat-Kinase (EC 2.7.2.3) nach Stärkegelektrophorese wird beschrieben. Das in der Vorwärtsreaktion des Enzyms gebildete Adenosin-Triphosphat wird mit einem Indikatorsystem (Hexokinase, Glukose-6-phosphat-Dehydrogenase, Tetrazoliumsalz) erfasst.

IN VIVO RED CELL GLYCOLYTIC CONTROL AND DPG-ATP LEVELS *

Much of clinical medicine involves a struggle against hypoxic diseases (such as heart disease, pulmonary disease, and anemia), all of which owe their morbidity and mortality ultimately to inadequate tissue oxygenation. Because of the quantitative significance of these kinds of diseases in health today, it is particularly important to try to understand and exploit the possible compensatory mechanisms for inadequate tissue oxygenation. FIGURE 1 puts the role of the red cell in perspective with other factors influencing tissue oxygenation. As the figure indicates, the factors that influence tissue oxygenation can be considered under four categories : cardiac output, pulmonary oxygen exchange, red cell oxygen transport, and lastly, hematological parameters, that is, red cell mass and amount of hemoglobin per red cell. Of course, an abnormality in any one of the four oxygen transport factors can be compensated somewhat by alterations in the other three factors. For instance, anemia leads to an increase in cardiac output and hyperventilation as well as to changes in red cell oxygen transport. Our emphasis in this paper, however, centers on those changes within the individual red cell that may improve its oxygen delivery during each transit through the tissue. Potentially, the most important change in this regard is a decrease in the oxygen affinity of the red cell (a shift to the right of the oxygen dissociation curve). A question of importance that has not yet been answered is whether or not oxygen affinity within the ranges seen normally and pathologically in various mammalian species really influences function. Theoretically, it can be shown in man that a 3 mm right shift in the oxygen dissociation curve, as might be produced by a 25% increase in DPG,t has a potential for 22% greater oxygen transport if cardiac output, ventilation, and hemoglobin levels remain the same.’ This theoretical benefit is based upon essentially a resting situation in which blood with a normal oxygen affinity returns to the lungs with approximately 70% of its oxygen remaining, whereas with a decrease in oxygen affinity the blood returns more deoxygenated. With stress such as exercise or in certain organs such as the heart, desaturation is more complete and the theoretical benefit of decreased oxygen affinity is relatively less. The effect of changes in oxygen affinity can also be examined in terms of the hypothetical drop in tissue

Enzymatic Mechanisms of Red Cell Adaptation to Anemia

It has long been known that many anemic patients adjust to their low hemoglobin levels and tend to have clinical symptoms less severe than might be expected. It is now generally accepted that the increased level of DPG in red blood cells of anemic patients plays a major role in this adjustment through its right- shifting effect on the erythrocyte-oxygen affinity curve. The biochemical mechanisms by which DPG levels are increased in anemia have yet to be satisfactorily demonstrated. It is known that DPG is produced by a two enzyme shunt in the glycolytic pathway (Rapoport and Luebering, 1950). The DPG shunt bypasses an ATP producing enzyme, phosphoglycerate kinase. There are three possible mechanisms by which DPG levels may be elevated: The first, a block in the lower part of the pathway, is trivial in our considerations here because it leads to decreased glycolysis and to hemolytic anemia if the block is severe enough. The second mechanism for elevating DPG levels would be an increase in the DPG shunt activity relative to the phosphoglycerate kinase step. It seems unlikely that this is the predominant mechanism because this implies that there should be a tendency for ATP levels to be low in anemia and this is not the case in most types of anemia.

Potential Effects of Hemoglobin Concentration on Red Cell Metabolism Together with Observations on Red Cell Metabolic Differences Between Men and Women

One of our major interests has been the glycolytic control mechanisms in the red cell which determine DPG levels normally and during hypoxic stress. We have increasingly utilized the method of assaying the measurable intermediates throughout the glycolytic pathway, along the lines suggested by Yoshikawa and Minakami (1968), in order to determine the key enzymatic step or steps which bring about changes in DPG. Through these studies we have begun to discern a rather consistent pattern, suggesting that the activity of hexokinase, already known to be one of the key regulators of erythrocyte glycolysis (Rapoport, 1968; Yoshikawa and Minakami, 1968), may be a very important factor in regulating DPG levels, in a number of circumstances. This pattern is illustrated in papers on anemia (Oelshlegel, et al. 1972) and altitude adaptation (Moore, et al. 1972) in this volume, but we would also like to illustrate it in the first part of this paper in differences between normal men and women.

Red Cell Metabolic Changes in Acute and Chronic Exposure to High Altitude

The red blood cell has occupied a central position in studies of how man adapts to high altitudes. With the discovery that the level of DPG has important implications for erythrocyte oxygen affinity, the study of red cell metabolism at high altitudes has taken on new and greater significance. Change in the level of DPG is most certainly related to changes in the levels of other red cell glycolytic intermediates. The appropriate next steps in understanding the mechanism of the increase in DPG are to ascertain which of the enzymes of glycolysis are activated by altitude exposure, and to identify the stimuli which are operating on this enzyme or enzymes. We initiated this next step with studies in Leadville, Colorado, elevation 10,200 feet.

Association between cholesterol and 2,3-diphosphoglycerate in genetically selected hooded rat lines.

We have developed two strains of hooded rats with differing erythrocyte oxygen affinities by selection on red cell 2,3-diphosphoglycerate levels. Genetic studies have shown that these strains differ at one DPG-level-determining locus. This article reports the results of a study which involved measurement of plasma cholesterol levels in rats from the strains and the F2 progeny of strain intercrosses. Low-DPG strain rats, with high oxygen affinity, had significantly higher mean cholesterol levels than High-DPG rats. Animals from the extremes of the F2 distribution of DPG levels showed similar, significantly different mean cholesterol levels, indicating that the negative association between DPG and cholesterol levels in strain rats was not due to inadvertent fixation of unrelated genes during selection on DPG. The possibility is discussed that high oxygen affinity, brought about by low DPG levels, may be causative in increasing cholesterol levels.

Interspecific variation of phosphoglycerate kinase in drosophila

The phosphoglycerate kinase electrophoretic banding patterns of several Drosophila species were studied. 2. Three banding types were noted: A type with a slowly migrating band of activity, a type with a faster migrating band of activity, and a type with an intermediate migrating band. 3. These banding types appeared to be species specific and may be useful in evolutionary or tax- onomical studies.