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698 Part VI: The Erythrocyte Chapter 47: Erythrocyte Enzyme Disorders 699

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the circulation red cells normally rely on glucose as their energy source, enzymes, for example, PK. In the process of reducing peroxides or
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the use of other substrates, particularly during blood storage (Chap. oxidized protein sulfhydryl groups, GSH is converted to GSSG, or may
138) and in certain experimental situations, is of interest. form mixed disulfides. Thus, for instance, GSSG has the capacity to
Glutathione Metabolism of the Erythrocyte The red cell con- inhibit red cell HK, 49,206 although greater than physiologic levels may be
tains a high concentration of the sulfhydryl-containing tripeptide GSH. needed for this effect. It may also complex with hemoglobin A to form
It serves a major role in antioxidant defense, detoxification, and main- hemoglobin A . 3 207
tenance of thiol status. Reported concentrations range between 0.4 and Glutathione reductase (GR) provides an efficient mechanism
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3.0 mM, with a terminal half-life (T ) of approximately 4 days. The for the reduction of GSSG to regenerate GSH, and thus maintain-
1/2
wide interindividual range suggests that GSH levels are, at least in part, ing high intracellular levels of GSH. A mitochondrial and a cytoplas-
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genetically determined. In its role of defense against oxidative stress mic isozyme are both produced from the same mRNA, most likely by
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GSH is oxidized to glutathione disulfide (GSSG), which can be reverted alternative initiation of translation. GR is a homodimer, linked by a
back to GSH by mediation of glutathione reductase. In addition GSSG disulfide bridge. Each 56-kDa subunit contains four domains, of which
can be transported out of the cell. domains 1 and 2 bind FAD (flavin adenine dinucleotide) and NADPH,
Glutathione biosynthesis occurs in two ATP-dependent steps: respectively, with domain 4 constituting the interface. The protein
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is encoded by the GR gene, located on chromosome 8p21.1. GR spans
Glutamate + Cysteine + ATP → γ-Glutamylcysteine + ADP + Pi 50 kb, contains 13 exons, and encodes a 522-amino-acids-long protein.
γ-Glutamylcysteine + Glycine + ATP → GSH + ADP + Pi
GR is a flavin enzyme, and either NADPH or NADH may serve as a
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The first step is rate limiting and catalyzed by glutamate cysteine hydrogen donor. In the intact cell, only the NADPH system appears
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ligase (GCL) (γ-glutamylcysteine synthetase). Feedback inhibition of to function. The same enzyme system appears to have the capacity to
42
GCL by GSH is commonly considered a key regulatory step in GSH reduce mixed disulfides of GSH and proteins. The activity of red cell GR
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homeostasis, but other pathways are likely to play a role in maintaining is strongly influenced by the riboflavin content of the diet, and its activ-
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GSH levels. GCL is a heterodimer composed of a catalytic 73-kDa ity is used as a biomarker for riboflavin status. Correction of partial GR
catalytic subunit (GCLC) and a 31-kDa modifier subunit (GCLM). 190,191 deficiency by riboflavin administration has been reported to ameliorate
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An intersubunit disulfide bond has been implicated in stabilization and hemolysis in a case of unstable hemoglobin. Hereditary deficiency of
catalytic efficiency of the heterodimer, acting as a cellular redox switch GR is a very rare disorder and associated with hemolytic anemia.
to couple enzyme activity, and consequently GSH levels, with the reduc- Red cells also contain thioltransferase that can catalyze GSH-
tion-oxidation (redox) state of the cell. 192,193 An alternative model sug- dependent reduction of some disulfides. 215,216
gests that increased levels of oxidative stress induce the formation of Oxidized glutathione is actively extruded from the erythro-
high activity heterodimer complexes from low activity monomeric and cyte. 217,218 GSSG efflux is likely an important regulator of GSH turnover
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holoenzyme forms of the enzyme. GCL subunits are encoded by sep- in red blood cells. The system consists of at least two GSSG-acti-
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arate genes, located on chromosome 6p12 (GCLC) and 1p22.1 (GCLM), vated adenosine triphosphatases (ATPases) that serve as an enzymatic
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respectively. GCLC contains 16 exons, encoding for the 637-amino- basis for this transport process. In addition to transporting GSSG,
acids catalytic subunit, whereas GCLM contains 7 exons that encode the the system appears to have the capacity to transport thioether conju-
274 amino acids of the modifier subunit. A trinucleotide repeat poly- gates of GSH and electrophiles formed by the action of glutathione-S-
morphism in the 5′-UTR affects GCLC gene expression and is associ- transferase. 221,222 Preliminary evidence indicates that multidrug
ated with alterations in GCL activity and GSH levels. GCL deficiency resistance protein 1 (MRP1) may be the exporter protein of both
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is a very rare cause of hemolytic anemia. glutathione-S conjugates and GSSG. 189,223 Erythrocytes contain gluta-
The second step in GSH synthesis is irreversible and mediated by thione-S-transferases rho, sigma, and theta. 224–226 Glutathione-S-trans-
glutathione synthetase (GS). This enzyme is a homodimer composed ferase catalyzes the formation of a thioether bond between GSH and
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of 52-kDa subunits, and the crystal structure of human GS has been a variety of xenobiotics. The role of glutathione-S-transferase in the
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resolved. There is one gene coding for GS. This 23-kb gene (GSS) is erythrocyte has not been established. It may be that it serves to cleanse
located on chromosome 20q11.2 and contains 13 exons, of which the the blood of xenobiotics to which the red cell membrane is permeable.
first is noncoding, that code for the 474-amino-acids-long protein. Defi- Glutathione-S-transferase could conjugate such substances to glutathi-
ciency of GS is the most common disorder of GSH synthesis and is asso- one, and the detoxified product of conjugation would be transported
ciated with hemolytic anemia. out of the red cell for subsequent disposal. The enzyme has the capacity
One important function of GSH in the erythrocyte is the detoxifi- to reversibly bind heme, and a possible role in heme transport has been
227
cation of low levels of hydrogen peroxide that may form spontaneously postulated. Deficiency of this enzyme is associated with hemolytic
or as a result of drug administration. Hydrogen peroxide is reduced anemia, but a cause-and-effect relationship has not been established. 228
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to water through mediation of the enzyme glutathione peroxidase, Other Antioxidant Enzymes Superoxide radicals that are formed
thereby oxidizing GSH to GSSG (see Fig. 47–7). Several glutathione are converted to hydrogen peroxide by the action of the copper-
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peroxidases exist but only type 1 appears to be expressed in red blood containing enzyme superoxide dismutase (SOD). Red blood cells
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cells. Glutathione peroxidase is a selenium-containing tetrameric contain SOD type 1 (SOD1). Mutations in SOD1 are associated with
200
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enzyme consisting of 21-kDa subunits. A polymorphism affecting the the dominant disorder familial amyotrophic lateral sclerosis. Patients
activity of this enzyme, which is most common in persons of Medi- display no hematologic phenotype. In agreement with this, studies on
terranean descent, has been described. The consequent decreases in SOD1-deficient mice have shown that a 50 percent reduced SOD activity
201
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enzyme activity are without clinical effect. In agreement with this, in red cells is probably sufficient to exert its protective effects. SOD1
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complete loss of glutathione peroxidase activity in mice was found to be null mice are viable, but show elevated levels of oxidative stress that
without any consequences, even at high levels of oxidative stress, thus causes regenerative anemia and triggers autoantibody production. 232
suggesting that glutathione peroxidase is of minor significance for red Hydrogen peroxide can be also decomposed to water and oxygen
cell function. 199 by catalase and peroxiredoxin. Both enzymes are abundantly present
GSH also functions in maintaining integrity of the erythrocyte in the red blood cell, suggesting an important role in the cell’s oxida-
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by reducing sulfhydryl groups of membrane proteins, and glycolytic tive defense. Nevertheless, acatalasemia, or catalase deficiency, is a
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