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

IV. Mature RBC
197
inorganic phosphate (P i ) being the most influential activators.
ADP is a less potent activator ( Jacobasch et al. , 1974 ).
Most of the adenine nucleotide pool in RBCs is maintained
as ATP under normal conditions, with less as ADP and
even less as AMP ( Tables 7-4 and 7-5 ). Glycolysis is ultimately
controlled by the demand for production of ATP.
As ATP is utilized, concentrations of ADP, AMP, and P i
increase. These changes result in the activation of PFK and
increased EMP metabolism. Other potential activators of
PFK include ammonium ions ( Debski and Rynca, 1985 ;
Shimizu et al. , 1988 ), glucose 1,6-bisphosphate ( Accorsi
et al. , 1985 ; Harvey et al. , 1992b ), fructose 2,6-bisphosphate
( Gallego and Carreras, 1990 ; Harvey et al. , 1992b ; Sobrino
et al. , 1987 ), and K (Ogawa et al. , 2002 ).
When PFK is activated, HK is activated secondarily
because the concentration of G6P is reduced, and G6P competitively
inhibits HK ( Rapoport et al. , 1976 ). Although the
HK reaction is normally not a rate-controlling step in glycolysis
of human RBCs, conceivably it is more important
in animal species with lower HK activity ( Rapoport, 1968 ).
The glycolytic rate is correlated with HK activity when
RBCs of various species are compared at pH values above
8.0. Comparisons may be less reliable when measurements
are made at pH 7.4 ( Harvey and Kaneko, 1976a ). In human
RBCs, the PK reaction becomes limiting when the PFK
reaction is markedly stimulated (i.e., at pH values above
7.6) ( Jacobasch et al. , 1974 ). The pH values listed earlier are
external values. The pH within RBCs is generally about 0.2
units lower than the external pH ( Waddell and Bates, 1969 ).
In addition to ATP/ADP and ADP/AMP ratios, various
other factors influence EMP metabolism. Alterations in
pH of plasma or in vitro buffers affect glycolysis. As pH is
increased above 7.2, PFK is activated and glucose utilization
and EMP metabolism increase ( Burr, 1972 ; Rapoport, 1968 ).
At physiological pH values, high concentrations of P i
stimulate glycolysis through the EMP by reducing the ATP
inhibition of PFK. Conversely, glycolysis is inhibited by
short-term phosphate deficiency, primarily by decreasing
intracellular P i for glyceraldehyde-3-phosphate dehydrogenase
(GAPD) ( Jacobasch et al. , 1974 ; Ogawa et al. , 1989 ;
Wang et al. , 1985 ). Decreased glycolytic rates result in
decreased RBC ATP concentrations and hemolytic anemia
in experimental dogs made severely hypophosphatemic by
hyperalimentation ( Jacob et al. , 1973 ; Yawata et al. , 1974 ).
Hemolytic anemia associated with hypophosphatemia has
also been reported in diabetic cats and a diabetic dog following
insulin therapy ( Adams et al. , 1993 ; Perman and
Schall, 1983 ; Willard et al. , 1987 ), in a cat with hepatic
lipidosis ( Adams et al. , 1993 ), and in postparturient cattle
in which decreased RBC ATP concentrations have been
measured ( Ogawa et al. , 1987, 1989 ). In addition to having
low ATP concentrations, dog RBCs might hemolyze as
a result of decreased RBC 2,3DPG concentration, because
dog RBCs with low 2,3DPG are more alkaline fragile than
those of normal dogs and may hemolyze at physiological
pH values ( Harvey et al. , 1988 ).
Several glycolytic enzymes, including PFK, GAPD, and
aldolase, bind to the cytoplasmic domain of band 3, forming
multimeric complexes on the inner RBC membrane. Other
enzymes, including PK and lactate dehydrogenase (LDH),
are associated with these complexes but do not directly
bind to band 3 ( Campanella et al. , 2005 ; Chu and Low,
2006 ). The binding of enzymes to the cytoplasmic domain
of band 3 results in their inhibition, which presumably also
has an inhibitory effect on RBC glycolysis ( Weber et al. ,
2004 ). The assembly of these glycolytic enzyme complexes
appears to be regulated by band 3 phosphorylation and Hb
oxygenation. The phosphorylation of tyrosine in the cytoplasmic
domain of band 3 by protein tyrosine kinases prevents
the binding of glycolytic enzymes, which presumably
enhances glycolysis ( Campanella et al. , 2005 ). The deoxygenation
of RBCs also dislodges the glycolytic enzymes
from the membrane, consistent with the established ability
of DeoxyHb, but not OxyHb, to bind the NH 2 terminus of
the band 3, which may contribute to the increased glycolytic
rate present in deoxygenated RBCs compared to oxygenated
RBCs ( Weber et al. , 2004 ).
There is a strong positive correlation between intracellular
Mg 2 and ATP concentrations in RBCs from various
species because of the presence of the Mg 2 -ATP complex
within cells ( Miseta et al. , 1993 ). RBCs of rats and dogs
with short-term magnesium deficiency have lowered glycolytic
rates, because adenine nucleotide substrates in four
glycolytic kinase reactions (HK, PFK, PGK, and PK) must
be complexed with Mg 2 (Rapoport, 1968 ). Dogs and rats
on magnesium-deficient diets become anemic ( Elin and
Alling, 1978 ; Kruse et al. , 1933 ), owing to shortened RBC
life spans.
The saturation of Hb with oxygen has an effect on glucose
utilization. Human RBCs utilize more glucose when
incubated anaerobically under nitrogen than under aerobic
conditions ( Asakura et al. , 1966 ). OxyHb is a stronger acid
than DeoxyHb; consequently, the intracellular pH of human
RBCs is lower in oxygenated blood than in deoxygenated
blood ( Takano et al. , 1976 ). The PFK reaction is inhibited
as blood is oxygenated due to the pH effect. In human
RBCs, 2,3DPG is bound to DeoxyHb and released on oxygenation.
Based on studies of glycolytic intermediates, the
increased unbound 2,3DPG in oxygenated RBCs may have
additional inhibitory effects on glycolysis ( Hamasaki et al. ,
1970 ). The effect of oxygenation on glycolysis of RBCs
from domestic animals remains to be determined. It may
not be important in ruminants because oxygenation results
in insignificant decreases in intracellular pH values ( Takano
et al. , 1976 ). As discussed earlier, deoxygenation of RBCs
dislodges certain glycolytic enzymes from the cytoplasmic
domain of band 3, which may contribute to the increased
glycolytic rate present in deoxygenated RBCs compared to
oxygenated RBCs ( Weber et al. , 2004 ).
2,3DPG inhibits glycolysis in part because of its reduction
of intracellular pH as a consequence of the Donnan
effect of this nonpenetrating anion ( Duhm, 1975 ). 2,3DPG