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

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Chapter | 7 The Erythrocyte: Physiology, Metabolism, and Biochemical Disorders
also inhibits glycolysis by inhibiting HK, PFK, and PK in
a manner different from its pH effect on these enzymes
(Duhm, 1975 ; Jacobasch et al. , 1974 ). In addition, 2,3DPG
inhibits 6-phosphofructokinase-2-kinase, the enzyme
responsible for the synthesis of the positive PFK effector
fructose-2,6-bisphosphate ( Sobrino et al. , 1987 ).
The RBC glycolytic rate is higher in dogs with HK
RBCs compared to dogs with LK RBCs. Glycolysis was
stimulated by increasing intracellular K concentration in
both types of RBCs, at least in part by the activation of PFK
and PK by K (Ogawa et al. , 2002 ).
Glycolysis increases in human RBCs with increasing
temperature to a maximum at 45°C, with a Q 10 of 2
( Rapoport, 1968 ). Based on measurements of glycolytic
intermediates, the major effect of temperature appears to be
on the PFK reaction ( Jacobasch et al. , 1974 ).
H . Diphosphoglycerate Pathway
Molecules of 1,3-diphosphoglycerate (1,3DPG), produced
by the GAPD reaction, may be utilized by the PGK reaction
in the EMP or may be converted to 2,3DPG by the
diphosphoglycerate mutase (DPGM) reaction ( Fig. 7-5 ).
2,3DPG degradation to 3-phosphoglycerate (3PG) is catalyzed
by diphosphoglycerate phosphatase activity (DPGP).
A single protein is responsible for both the DPGM and
DPGP activities ( Sasaki et al. , 1977 ). The DPG pathway
or shunt (Rapoport-Luebering cycle) bypasses the ATPgenerating
PGK step in glycolysis; consequently, no net
ATP is generated when glucose is metabolized through this
pathway ( Brewer, 1974 ).
Normally, from 10% to 30% of triose phosphate metabolism
in human RBCs is shunted through this pathway
(Jacobasch et al. , 1974 ; Mulquiney et al. , 1999 ; Oxley et al. ,
1984 ). The proportion of 1,3DPG metabolized by PGK and
DPGM is determined mostly by the concentration of ADP
( Rapoport, 1968 ). ATP and 2,3DPG influence the relative
amount of flow through each route by product inhibition of
their own synthesis.
The absolute flow is also determined by the overall
glycolytic rate. 2,3DPG inhibits HK, PFK, and GAPD in
human RBCs ( Srivastava and Beutler, 1972 ). The formation
of 2,3DPG is stimulated by increased P i concentration and
increased pH, which stimulate glycolysis by activating PFK
greater than PK ( Jacobasch et al. , 1974 ). Hypoxic conditions
stimulate 2,3DPG synthesis primarily by inducing hyperventilation,
which results in alkalosis; however, intracellular pH
also increases in hypoxia because DeoxyHb binds more H
than OxyHb ( Duhm and Gerlach, 1971 ). Conversely, acidosis
and hypophosphatemia result in decreased 2,3-DPG concentrations
( Haglin et al. , 1994 ; Ibrahim et al. , 2005 ).
The concentration of 2,3DPG can be affected by a
decrease in PK activity. When PK activity is reduced relative
to PFK activity, as occurs in PK deficiency, phosphorylated
intermediates between the PK and GAPD reactions increase
in concentration ( Harvey, 2006 ; Mueggler and Black,
1982 ). The synthesis of 2,3DPG is increased as a result
of the increased concentration of the 1,3DPG substrate.
Thyroid hormones can increase 2,3DPG in human ( Snyder
et al. , 1970 ) and sheep ( Studzinski et al. , 1982 ) RBCs, possibly
by a direct effect on the DPGM enzyme.
RBCs of dogs, horses, pigs, and humans normally contain
high concentrations of 2,3DPG, whereas those of cats
and domestic ruminants have low concentrations ( Tables
7-4 and 7-5 ). Based on results from a large number of
mammalian species, only cats, hyenas, and civets among
carnivores, and deer, giraffe, antelope, and the cattle family
among artiodactyls have low RBC 2,3DPG concentrations
(Bunn, 1981 ; Bunn et al. , 1974 ). Low concentrations of
2,3DPG in cat, goat, and sheep RBCs result primarily from
low RBC DPGM activities, whereas RBCs of cattle have
relatively high DPGP activity in association with moderately
low DPGM activity ( Chemtob et al. , 1980 ; Harkness
et al. , 1969 ; Pons et al. , 1985 ).
The concentration of 2,3DPG increases more than twofold
during reticulocyte maturation in rats and rabbits, which
are species that naturally have high 2,3DPG concentrations
in their RBCs ( Gallego et al. , 1991 ). In contrast to these
species, sheep reticulocytes have low 2,3DPG concentration,
which decreases even further during reticulocyte maturation
to mature RBCs ( Gallego and Carreras, 1990 ).
I . Hb Oxygen Affinity
1 . Oxygen Dissociation Curve
DeoxyHb exists in a low affinity “ tense ” structure. With
oxygenation it undergoes a transition to OxyHb with a
“ relaxed ” structure that has a lower binding capacity for
CO 2 , H , Cl , and organophosphates ( Barvitenko et al. ,
2005 ). The initial binding of a molecule of O 2 to a monomer
of tetrameric, deoxygenated Hb facilitates further
binding of additional O 2 molecules to the Hb molecule.
Because the O 2 binding of one heme group influences the
affinity of other heme groups for O 2 , this characteristic has
been called the heme-heme interaction. The changing oxygen
affinity of Hb with oxygenation results in a sigmoid
oxygen dissociation curve ( Fig. 7-6 ) when the percentage
saturation of Hb with oxygen is plotted against the partial
pressure of oxygen (pO 2 ). The pO 2 at which Hb is 50%
saturated is the P 50 . The steepness of the middle portion
of the curve is of great physiological significance, because
it covers the range of oxygen tensions present in tissues.
Consequently, relatively small decreases in oxygen tension
can result in substantial oxygen release from Hb ( Benesch
et al. , 1975 ).
When blood from many mammalian species is studied,
an inverse linear correlation is recognized between the log
P 50 of whole blood and the log of body weight ( Scott et al. ,