Samuel M. Rapoport

Chapter 3 Hemolytic anemias due to erythrocyte enzyme deficiencies

Abstract Red blood cells can only fulfil their functions over the normal period of approximately 120 days with 1.7 × 105 circulatory cycles efficiently if they withstand external and internal loads. This requires ATP and redox equivalents, which have to be permanently regenerated by the energy and redox metabolism. These pathways are neccessary to maintain the biconcave shape of the cells, their specific intracellular cation concentrations, the reduced state of hemoglobin with a divalent iron and the sulfhydryl groups of enzymes, glutathione and membrane components. If an enzyme deficiency of one of these metabolic pathways limits the ATP and/or NADPH production, distinct membrane alterations result causing a removal of the damaged cells by the monocyte-macrophage system. Most metabolic needs of erythrocytes are covered by glycolysis, the oxidative pentose phosphate pathway (OPPP), the glutathione cycle, nucleotide metabolism and MetHb reductase. Hereditary enzyme deficiencies of all these pathways have been identified; those that cause non-spherocytic hemolytic anemia are listed in Table 4. Their frequencies differ markedly both with respect to the affected enzyme and geographic distribution. Glucose-6-phosphate dehydrogenase enzymopathies (G6PD) are with more than 400 million cases by far the most common deficiency. The highest gene frequency has been found with 0.7 among Kurdish Jews. G6PD deficiencies are furthermore prevalent with frequencies of about 0.1 among Africans, Black Americans, and populations of Mediterranean countries and South East Asia. In Middle and Northern Europe the frequency of G6PD is much lower, and with approximately 0.0005, comparable with the frequency of pyruvate kinase (PK) enzymopathies, the most frequent enzyme deficiency in glycolysis in this area (Luzzatto, 1987; Beutler and Kuhl, 1990). The relationship between the degree of enzyme deficiency and the extent of metabolic dysfunction in red blood cells and other tissues depend on several factors: on the importance of the affected enzyme; its expression rate; the stability of the mutant enzyme against proteolytic degradation and functional abnormalities; the possibility to compensate the deficiency by an overexpression of the corresponding isoenzyme or by the use of an alternative metabolic pathway. Difficulties in estimating the quantitative degree of disorder in severe cases are due to the fact that these populations contain many reticulocytes, which generally have higher enzyme activities and concentrations of intermediates than erythrocytes. An alternative approach to predict metabolic changes is the analysis by mathematical modeling. Mathematical modeling of the main metabolic pathways of human erythrocytes has reached an advanced level (Rapoport et al., 1976; Holzhutter et al., 1985; Schuster et al., 1988). Models have been succesfully employed to describe stationary and time-dependent metabolic states of the cell under normal conditions as well as in the presence of enzyme deficiencies. Figure 5 shows computational results of erythrocyte enzyme deficiencies. This analysis is based on the comprehensive mathematical model of the energy and redox metabolism for human erythrocyte presented in Fig. 6. Stationary states of the cell metabolism have been calculated by varying the activity of each of the participating enzymes by several orders of magnitude. To predict consequences of enzyme deficiencies a performance function has been introduced (Schuster and Holzhutter, 1995). It takes into account the homeostasis of three essential metabolic variables: the energetic state (ATP), the reductive capacity (reduced glutathione) and the osmotic state. From the data given in Fig. 5 one can conclude that generally the metabolic impairment resulting in deficiences occurs earlier for enzymes with high control coefficients than for those catalyzing equilibrium reactions. On the other hand the flux curves of latter enzymes decrease more steeply below a critical threshold. Furthermore, one can estimate the range of enzyme activities in which the metabolic alterations should be either tolerable or associated with non-chronic or chronic hemolytic anemia. Enzymes responsible for maintenance of glutathione in its reduced state have a wide range of activity where hemolytic crises are expected only under stress conditions, whereas most enzymopathies of glycolysis lead to chronic hemolytic anemia.

Proteolysis of mitochondria in reticulocytes during maturation is ubiquitin-dependent and is accompanied by a high rate of ATP hydrolysis

The ATP‐dependent breakdown of mitochondria‐containing stroma proceeds via the ubiquitin‐requiring pathway. The proteolysis is linked to a large ATP‐cleaved consumption amounting to 1 ATP per peptide bond or more. Proteins of mitochondria‐containing stroma are much better substrates of ATP‐ubiquitindependent proteolysis than heat‐denatured ones. Hemin suppresses both proteolysis and ATP hydrolysis.

Maturational Breakdown of Mitochondria and Other Organelles in Reticulocytes

One of the characteristics of the differentiation of erythroid cells is the decay or elimination of organelles, including the nucleus, mitochondria, ribosomes, lysosomes, endoplasmic reticulum, and Golgi apparatus. Many of the changes occur in the nucleated precursors of the erythrocyte. Some organelles, however, primarily mitochondria and ribosomes, but also vestiges of others, remain in the reticulocyte. The mechanisms involved in the degradation of organelles are largely unexplored. The process best understood is the maturational breakdown of mitochondria in reticulocytes, which will therefore be the focus of the present review. The various changes appear to constitute a fixed program of maturation that once started takes it course with little or no outside effectors. The interplay of the various events and their causal relationships are open questions.

Oxygenation of biomembranes by mammalian lipoxygenases: the role of ubiquinone.

15-Lipoxygenase is implicated in the selective breakdown of mitochondria during red cell maturation by virtue of its capability of directly oxygenating phospholipids. To address the reason of the selectivity for mitochondria, we studied the reaction of pure rabbit 15-lipoxygenase with beef heart submitochondrial particles in vitro. This reaction is characterised by a loss of polyenoic fatty acids, the formation of phospholipid-bound hydroperoxy- and keto-polyenoic fatty acids, and oxidative modification of membrane proteins. The total oxygen uptake exceeds the formation of oxygenated polyenoic fatty acids several times. The excessive oxygen uptake was not inhibited by 3,5-di-tert-butyl-4-hydroxytoluene or by respiratory inhibitors, but was partly suppressed by superoxide dismutase plus catalase, salicylate, or mannitol. Pentane-extraction of the submitochondrial particles abolished the excessive oxygen uptake, whereas reconstitution with ubiquinone- 50 restored it. A marked excessive oxygen uptake did not occur during the analogous reaction with erythrocyte ghosts. It is proposed that ubiquinone-50 triggers the formation of hydroxyl radicals from 15-lipoxygenase-derived hydroperoxy-lipids via a Fenton-type reaction driven by ubisemiquinone radicals. A new prooxidative function of ubiquinone in the biologically programmed degradation of mitochondria in certain types of cells is proposed.

Maturation of rabbit reticulocytes: susceptibility of mitochondria to ATP-dependent proteolysis is determined by the maturational state of reticulocyte

(1) A simple procedure is described to separate reticulocytes of different maturity in high yield. (2) It is shown that exhaustion of supply of mitochondria susceptible to degradation by the lipoxygenase‐ATP‐dependent proteolysis system limits the extent of breakdown of mitochondria during in vitro maturation. The susceptibility of mitochondria depends on the maturity of the reticulocytes. (3) Incubation in the presence of calcium ions and calcium ionophore leads to full susceptibility of mitochondria in immature reticulocytes but has no effect on those in mature reticulocytes which are already fully susceptible to degradation. (4) Conditions which lead to rapid degradation of mitochondria do not affect the behaviour of the reticulocyte count. There appears to be no obligatory connection between the breakdown of mitochondria and of ribosomes.

Pentane formation during the anaerobic reactions of reticulocyte lipoxygenase. Comparison with lipoxygenases from soybeans and green pea seeds.

The lipoxygenases from reticulocytes, soybeans and green pea seeds produce pentane in an anaerobic assay containing 13Ls-hydroperoxy-9-cis, 11-trans-octadecadienoic acid and 9,12-all-cis-octadecadienoic acid. The presence of oxygen strongly inhibits pentane formation by the three enzymes. Relative to the lipoxygenase activity with linoleic acid as substrate, soybean lipoxygenase is 4-times as effective in pentane formation as the lipoxygenases from reticulocytes or green pea seeds. Pentane formation by the reticulocyte lipoxygenase is completely inhibited by lipoxygenase inhibitors (5,8,11,14-eicosatetraynoic acid, 3-t-butyl-4-hydroxyanisol) but only partially by radical scavengers which do not influence the oxygenase activity (2,6-di-t-butyl-4-hydroxytoluene). From the temperature dependence below 20 degrees C an activation energy of the pentane production by the reticulocyte lipoxygenase of about 28 kJ/mol was calculated, which is somewhat higher than that for the oxygenase activity. During the anaerobic reaction of both reticulocyte and soybean lipoxygenase C18-oxodienes, C13-oxodienes, linoleic acid dimers and a polar compound proposed to be epoxy-hydroxyoctadecenoic acid are produced in a similar pattern. Reticulocyte lipoxygenase produces pentane with submitochondrial particles only under anaerobic conditions after an aerobic preincubation. During the incubation of intact reticulocytes with the calcium ionophore A23187 or arachidonic acid, pentane is released. Preincubation of the cells with lipoxygenase inhibitors completely abolishes the pentane formation. Erythrocytes do not form any pentane under the same experimental conditions.

Reticulocyte lipoxygenase exhibits both n-6 and n-9 activities

Purified reticulocyte lipoxygenase converts arachidonic acid to both 15‐ and 12‐hydroxyperoxyeicosatetraenoic acids. The proportion of the two reaction products does not change during the purification procedure as shown by HPLC analysis. By means of isoelectric focusing it was not possible to separate the n‐6 and n‐9 activities. Reticulocyte lipoxygenase was completely inactivated by both 5,8,11‐eicosatriynoic and 5,8,11,14‐eicosatetraynoic acids in contrast to soybean lipoxygenase‐1 which inactivated only by 5,8,11,14‐eicosatetraynoic acid. These results indicate that reticulocyte lipoxygenase exhibits both n‐6 and n‐9 activities. A contamination of the enzyme preparation with other lipoxygenases, e.g., the n‐9 lipoxygenase from thrombocytes appears to be excluded.

Characteristics of an ATP-dependent proteolytic system of rat liver mitochondria.

Rat liver mitochondria contain an ATP‐dependent proteolytic system which is localized on the outside of the inner membrane. It is capable of utilizating both the ATP produced within the mitochondria as well as that supplied externally. The system is dependent on Ca2+. Its physiological function is seen in the normal breakdown of mitochondria during their turnover. The system may be selective for the breakdown of the inner membranes.

Response of the glycolysis of human erythrocytes to the transition from the oxygenated to the deoxygenated state at constant intracellular pH

The time course of the rate of the glycolysis of human erythrocytes and of some metabolites were determined before and after rapid deoxygenation at constant intracellular pH. For this purpose stripped deoxygenated haemoglobin was used as a rapid oxygen acceptor. Deoxygenation causes an increase of the glycolytic rate by 26%. Glucose 6-phosphate is decreased while the adenine nucleotides and 2,3-bisphosphoglycerate remain constant. Fructose 1,6-bisphosphate and the triose phosphates decrease transiently before rising. The data can be explained by increased binding of phosphocompounds to deoxygenated as compared with oxygenated haemoglobin. Thereby the control enzymes hexokinase and phosphofructokinase are influenced. It is concluded that under physiological conditions changes in the oxygenation state of haemoglobin per se alter the glycolytic rate.

Reversibility of the inhibition of cytochrome c oxidase by reticulocyte lipoxygenase.

During the maturation of reticulocytes the mitochondria are lyzed and inactivated by a cell-specific lipoxygenase (LOX) [ 11. In addition to oxygenation of mitochondrial phospholipids the LOX action gives rise to an inhibition of the respiratory chain at three sites. Two inhibitory sites are located between the Fe-S centers of complex I and complex II and ubiquinone [2]. The third inhibitory site located at the cytochrome c oxidase differs from the others with respect to the kinetics, dose-dependence and the extent of the inhibition [3,4]. The present paper deals with the mechanism of the inhibitory action of reticulocyte LOX on particulate cytochrome c oxidase. As reported elsewhere, even extensive LOX-treatment of submitochondrial particles (SMP) does not affect the amount and the spectral properties of the redox-active components of the cytochrome c oxidase [ 1,2], which rules out the possibility of co-oxidative destruction of these components. In this paper evidence will be adduced that LOX inhibits cytochrome c oxidase by chemical modification of the membrane phospholipids which are essential for its activity.