Clinical Biochemistry of Domestic Animals (Sixth Edition) - UMK ...

III. Developing Erythroid Cells
181
α -globin genes, based on their location in the α-globin gene
cluster. Gene expression progressively decreases from 5
to 3 (Vestri et al. , 1994 ). Compared to sheep with duplicated
α -globin genes, sheep with triplicated α-globin genes
produce excess α -globin, resulting in an unbalanced alpha/
beta ratio and larger RBCs with increased osmotic fragility
(Pieragostini et al. , 2003 ).
Considerable heterogeneity of Hb types occurs in adult
animals. Two or more Hb types are reported to occur in
domestic animal species ( Braend, 1988 ; Kitchen, 1974 ;
Kohn et al. , 1998, 1999 ; Pirastru et al. , 2003 ; Rando and
Masina, 1985 ). Most polymorphism of animal Hbs is determined
genetically and is usually caused by multiple amino
acid interchanges ( Kitchen, 1974 ). Hb types were initially
classified based on electrophoretic mobility in gels.
Electrophoretic differences can result from differences in α
or β chain structure. For example, HbA and HbB in sheep are
attributable to differences in β chains ( Garner and Lingrel,
1988 ), but HbA and HbB in goats are attributed to differences
in α chains ( Huisman and Kitchens, 1968 ; Pieragostini
et al. , 2005 ). Electrophoretic mobility in gels is not sensitive
enough to identify many α and β chain variants, and distinctly
different Hb tetramers can migrate with similar mobilities,
resulting in misleading information. For example, HbB
( α 2
B
β 2
A
) and HbD ( α 2
A
β 2
D
) in goats have almost the same
mobility at alkaline pH ( Pieragostini et al. , 2005 ).
Nongenetic alterations in Hb structure can also contribute
to apparent Hb heterogeneity. Examples include the N-
acetylation of beta chains in cat HbB ( Taketa et al. , 1972 )
and glycosylation of Hb, a function of intracellular glucose
concentration and RBC life span ( Higgins et al. , 1982 ;
Rendell et al. , 1985 ). Increased glycosylation of Hb has been
reported in diabetic dogs and cats ( Elliott et al. , 1997, 1999 ).
Similar to the glycosylation of Hb by glucose, cyanate combines
spontaneously and irreversibly with Hb to form carbamylated
Hb. Cyanate forms spontaneously from urea. Dogs
with chronic renal failure and long-term increases in urea
concentration have larger amounts of carbamylated Hb than
dogs with acute renal failure ( Heiene et al. , 2001 ).
D . Reticulocytes
1 . Formation
Most reticulocytes are formed from metarubricytes within
the extravascular space of the bone marrow by a process
of nuclear extrusion that requires functional microtubules
(Chasis et al. , 1989 ) and is likened to mitosis ( Bessis, 1973 ;
Simpson and Kling, 1967 ). The erythroblast macrophage
protein (Emp) associates with F-actin and is important in
denucleation of metarubricytes, as well as blood island formation
( Soni et al. , 2006 ). Extruded nuclei quickly expose
phosphatidylserine on their surfaces, which promotes their
binding to, and phagocytosis by, blood island macrophages
(Yoshida et al. , 2005 ).
Reticulocyte cytoplasm contains ribosomes, polyribosomes,
and mitochondria necessary for the completion of
Hb synthesis. Reticulocytes derive their name from a network
or reticulum that appears when stained with basic
dyes such as methylene blue and brilliant cresyl green. That
network is not preexisting but is an artifact formed by the
precipitation of ribosomal ribonucleic acids and proteins.
As reticulocytes mature, the amount of ribosomal material
decreases until only a few basophilic specks can be visualized
with reticulocyte staining procedures. These mature
reticulocytes have been referred to as type IV ( Houwen,
1992 ) or punctate reticulocytes (Alsaker, 1977 ; Perkins
et al. , 1995 ). To reduce the chance that a staining artifact
would result in misclassifying a mature RBC as a punctate
reticulocyte using a reticulocyte stain, the cell being evaluated
should have two or more discrete blue granules that are
visible without requiring fine focus adjustment of the cell to
be classified as a punctate reticulocyte.
2 . Metabolism
Reticulocyte metabolism and maturation have been reviewed
by Rapoport (1986) and are summarized here. Immature
reticulocytes continue to synthesize protein (primarily globin
chains) with residual mRNA, tRNA, and rRNA formed
before denucleation. About 30% of total Hb is synthesized
after nuclear extrusion ( Geminard et al. , 2002 ). Synthesis of
fatty acids is minimal, but phospholipids are synthesized from
preformed fatty acids. Substrates for protein and lipid synthesis
and for energy metabolism are provided from endogenous
sources (breakdown of mitochondria and ribosomes) as well
as from plasma. The reticulocyte can synthesize adenine and
guanine nucleotides de novo (Rapoport, 1986 ).
Most ATP is generated in reticulocytes by oxidative
phosphorylation in mitochondria. Glucose is the major substrate,
but amino acids and fatty acids can also be utilized
for energy ( Rapoport, 1986 ).
3 . Maturation into RBCs
Reticulocyte maturation into mature RBCs is a gradual process
that requires a variable number of days depending on
the species involved. Consequently, the morphological and
physiological properties of reticulocytes vary with the stage
of maturation. Early reticulocytes have polylobulated surfaces.
The cell surface undergoes extensive remodeling with
loss of membrane material and ultimately the formation of
the biconcave shape of mature RBCs ( Bessis, 1973 ). The
loss of membrane protein and lipid components requires
ATP and involves the formation of intracellular multivesicular
endosomes that fuse with the plasma membrane releasing
vesicles (exosomes) extracellularly ( Geminard et al. , 2002 ).
This is a highly selective process in which some proteins
(e.g., TfR1 and fibronectin receptor) are lost and cytoskeletal
proteins (e.g., spectrin) and firmly bound transmembrane