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74 Part I Molecular and Cellular Basis of Hematology

biochemical pathways to provide energy and building blocks for enzymatic complex. Acetyl-CoA is a high-energy intermediate
macromolecules that constitute the cell or regulatory metabolites. that can be further oxidized by the TCA cycle or utilized for fatty
Glucose can be stored in cells in the form of glycogen, which consti- acid synthesis. The TCA cycle is initiated by the condensation of
tutes a rapid source of energy through its breakdown to free glucose oxaloacetic acid with acetyl-CoA, forming citrate. In reactions involv-
(glycogenolysis), although this pathway is limited to a certain number ing decarboxylation and oxidation, CO 2 is produced and NADH
of hematopoietic cells. Chemotaxins (FMLP, C5ades arg, arachidonic and flavin adenine dinucleotide (FADH) are produced for use in
acid) activate granulocytes to catabolize significant amounts of the mitochondrial respiratory chain. The flux of the TCA cycle is
endogenous glycogen. regulated by the levels of acetyl-CoA and oxaloacetic acid, which are
+
+
entry points in the cycle, and by the availability of NAD and FAD
substrates. The rate of oxidation through the TCA cycle depends
Glycolysis on mitochondrial electron transport activity, which is governed in
part by NADH levels. The TCA cycle also produces metabolites for
Glycolysis is a series of reactions by which six-carbon glucose is biosynthetic processes (anaplerotic reactions). For example, citrate is
converted into two three-carbon keto-acids (pyruvate). Importantly, converted to fatty acids and sterols, and succinyl CoA is an intermedi-
these oxidative reactions generate energetic molecules such as ATP ate in heme and porphyrin synthesis. Aside from the bioenergetic
and NADH, and can occur in the absence of oxygen and mitochon- and anaplerotic aspect of this cycle, several reactions have important
dria. In some cells, such as erythrocytes, anaerobic glycolysis produces clinical implications.
lactate, but in most cell types pyruvate is completely oxidized to
acetyl coenzyme-A and carbon dioxide by the mitochondrial pyru-
vate dehydrogenase complex and the tricarboxylic acid (TCA) cycle Oxidative Phosphorylation
coupled to oxidative phosphorylation. In general, hematopoietic stem
cells are thought to largely depend on glycolysis, while more dif- In most cell types, oxidative phosphorylation is dominant on ATP
ferentiated cells, except for erythrocytes, use mitochondrial oxidative generation. Exceptions include red blood cells, which lack mito-
metabolism. Glycolytic fluxes are under intrinsically tight control chondria. Oxidative phosphorylation complexes are located at the
through intermediate metabolites in the pathway. The most powerful inner mitochondrial membrane and receive high-energy electrons
control is exerted by fructose 2,6-bisphosphate (F-2,6-BP), which is from NADH (produced from the oxidation of acetyl-CoA). These
generated by phosphofructokinase 2. F-2,6-BP allosterically activates electrons are passed through the different oxidative phosphoryla-
phosphofructokinase, providing a “feed-forward” mechanism of tion complexes (which contain heme, copper iron–sulfur groups,
stimulation. Activation of growth factor signaling pathways potently and flavins as electron carriers) until they reach the final electron
stimulate glycolysis at different points, including phosphorylation of acceptor, molecular oxygen. As a consequence of electron transfer,
phosphofructokinase 2 and pyruvate kinase. The PI3K pathway is a protons are pumped into the mitochondrial intermembrane space,
major signaling pathway that controls glycolysis. generating an electrochemical gradient used to synthesize ATP.
Interestingly, in erythrocytes, 1,3-diphosphoglycerate can be There are five oxidative phosphorylation complexes: complex I
diverted from glycolysis to synthesize 2,3-diphosphoglycerate (2,3- (NADH–CoQ reductase complex), complex II (succinate–CoQ
DPG) via the enzyme diphosphoglycerate (Rapoport–Laubering reductase complex), complex III (CoQH 2 –cytochrome c reductase
shunt). 2,3-DPG is an important metabolite that regulates oxygen complex), complex IV (cytochrome C oxidase complex), and
binding to hemoglobin; thus increased levels of 2,3-DPG (e.g., under complex V (ATP synthase complex). In general, hematopoietic
hypoxic conditions) allow hemoglobin to release oxygen under low stem cells are located in low-oxygen niches and largely depend on
partial oxygen tensions. glycolysis instead of oxidative phosphorylation to maintain ATP
levels. The differentiation process is associated with increases in
mitochondria, which allow for the generation of ATP through the
Pentose Phosphate Pathway respiratory chain. For example, this occurs in quiescent T cells that
are in a catabolic phase, producing ATP mainly through oxidative
The pentose phosphate pathway (PPP) derives from glycolysis in the phosphorylation. Upon stimulation, activated T cells shift towards
cytoplasm. The first enzyme in this pathway is glucose-6-phosphate an anabolic phase, relying upon a high rate of glycolysis for ATP
dehydrogenase (G6PDH) and produces NADPH, a substrate utilized generation. Mitochondrial DNA encodes for several oxidative
for lipogenesis and glutathione regeneration by glutathione reductase. phosphorylation subunits and mutations in this DNA produce
The regulation of NADPH production through G6PDH is through mitochondrial diseases. Interestingly, anemia, a symptom associated
NADPH-mediated product inhibition. The PPP is also important in with patients having Pearson syndrome, is caused by accumula-
generating ribose-5 phosphate, which is a precursor for nucleotide tion of mutated mitochondrial DNA in sideroblasts. This suggests
synthesis in proliferating cells. Interestingly, G6PDH deficiency leads that hematopoietic cell-specific respiration defects can be respon-
to low levels of NADPH, which is essential for controlling reactive sible for anemia by inducing abnormalities in erythropoiesis during
oxygen species (ROS) through glutathione reductase. It is one of development.
the most common erythrocyte enzymopathies and these cells cannot
prevent oxidative damage in critical molecules such as heme, causing
overall irreparable damage to the cell at a much higher rate than Reactive Oxygen Species Metabolism
normal, particularly in response to certain environmental triggers
such as drugs and stress. The damaged erythrocytes are removed from Reactive oxygen species (ROS) are chemically reactive small molecules
circulation in the spleen and destroyed by macrophages at an elevated with oxygen in different oxidation states, such as partially reduced
rate, leading to anemia. This enzymopathy occurs in areas with high oxygen ions and peroxides. The three major species are superoxide,
malarial burden, in part because the mutated recessive allele confers hydrogen peroxide, and hydroxyl radicals. The major cellular sites
malarial resistance. This resistance is because red blood cells with low for ROS production are the mitochondria and NADPH oxidase,
G6PDH activity, when infected with the parasite, are continuously a plasma membrane or phagosome-bound enzyme. Approximately
removed from the circulation. 85% of cellular ROS is a subproduct of normal oxidative phos-
phorylation. Superoxide is the initial ROS produced in the electron
transport chain, and it is transformed to hydrogen peroxide by the
Tricarboxylic Acid or Krebs Cycle enzyme superoxide dismutase. Hydrogen peroxide is the substrate
of catalase or glutathione peroxidase, which reduces it to water.
A major route for pyruvate oxidation is conversion to acetyl-CoA, Hydrogen peroxide, however, is also converted to hydroxyl radicals,
a reaction catalyzed by the mitochondrial pyruvate dehydrogenase the most reactive oxygen species, in a Fenton reaction with ferrous