CHAPTER 9

Energy Metabolism and Membrane Physiology of the Erythrocyte

RBC
transport oxygen and carbon dioxide
has sacrificed greatly for this purpose by losing its
nucleus
life span: 120 days

**********
ENERGY PRODUCTION ANAEROBIC
GLYCOLYSIS

when nucleus is ejected, so are the other organelles,
therefore no mitochondria
RBC then relies on glycolysis for energy production
oxygen exchange and binding to heme do not
require energy.

Hereditary nonspherocytic hemolytic anemia
shortened RBC survival due to deficiency in
enzymes involved in glycolysis

Cation pumps
transmembranous cation gradient proteins
ideal cation gradient: intracellular K+, Na
+
and
Ca
2+
, extracellular K+, Na
+
and Ca
2+
,
consume approximately 15% of RBC ATP
production

Energy enzymes degrade pumps fail Na
+
and Ca
2

and water rushes in cell swells cell destroyed

Facilitated membrane transport system
through which plasma glucose enters the RBC

Embden Meyerhof Pathway
anaerobic glycolysis
requires glucose to generate ATP
via which glucose is catabolized to pyruvate
consumes 2 ATP per glucose molecule, generates 4
ATP per glucose molecule; thus net gain is 2ATP

Glucose
high-energy phosphate source that is the greatest
reservoir of energy in the RBC

A. First phase
involves:
a. phosphorylation
b. isomerization
c. diphosphorylation
to yield fructose-1,6-bisphosphate (F-1,6-P)


F-1,6-P
substrate for aldolase cleavage for
glyceraldehyde-3-phosphate (G3P)

G3P
final product of phase I

Hexokinase and phosphofructokinase
rate-limiting in steady-state anaerobic glycolysis

Hexokinase
has the lowest activity of all the glycolytic
enzymes

B. Second phase
glucose-3-phosphate 3-phosphoglycerate
(G3P 3-PG)

C. Third phase
3-PG pyruvate

**********
GLYCOLYSIS DIVERSION PATHWAYS (SHUNTS)

A. Hexose Monophosphate Pathway
aerobic/oxidative glycolysis
aka pentose phosphate shunt
detoxifies accumulated peroxide
extends RBC functional life span by oxidizing
peroxide

peroxide
oxidizes heme iron, proteins and lipids
especially those containing thiol groups
arises spontaneously from the reduction of
oxygen in the cells aqueous environment

diverts G6P to pentose phosphate by action of
G6P dehydrogenase
oxidized NADP+ reduced to NADPH
NADPH reduces glutathione GSSG to GSH by
glutathione reductase

glutathione
sulfur-containing compound, tripeptide
containing cysteine

GSH then reduces peroxide to water and oxygen
via glutathione peroxidase
5% to 10% of G6P is diverted to the HMP
activity increases 20x to 30x after oxidative
challenge
oxidizes G6P at C1 to form ribulose-5-phosphate
only means of generating NADPH for glutathione
reduction
w/o it , RBC are vulnerable to oxidative damage

G6PD deficiency
most common human RBC enzyme deficiency

B. Methemoglobin Reductase Pathway
methemoglobin is formed when peroxide
oxidizes heme iron from Fe2+ to Fe3+
HMP can reduce peroxide, but it can do nothing
when methemoglobin has already been formed,
this is where methemoglobin reductase pathway
comes in
NADPH reduces methemoglobin in the presence
of methemoglobin reductase aka cytochrome
b5 reductase

cytochrome b5 reductase
acts as intermediate electron carrier
swiftly returns oxidized iron (Fe3+) to its
ferrous oxygen-carrying state (Fe2+)
accounts for 65% of the metHb reducing
capacity w/in the RBC

C. Rapoport Luebering Pathway
generates 2,3 bisphosphoglycerate (2,3-BPG)
from 1,3-bisphosphoglycerate (1,3-BPG) by
bisphosphoglycerate mutase (consumes 2ATP)

2,3-BPG
regulates oxygen delivery to tissues by
competing w/ oxygen for HB
oxygen is released when this binds, thus
oxygen is delivered to the tissues

2,3-BPG forms 3-PG via bisphosphoglycerate-
phosphatase
sacrifices the production of 2 ATP molecules via
glycolysis
cell is put in ATP deficit

bisphosphoglyceromutase
inhibited by acidic pH and low conc. of
3-PG and 2-PG (1,3-BPGis retained in the
EMP

bisphosphoglycerate phosphatase
activated by decreased
bisphosphoglyceromutase and ATP
returns 2,3-BPG to the mainstream of
glycolysis
generation of ATP is favored


**********
RBC MEMBRANE

RBC DEFORMABILITY

90 fL volume, 140m
2
surface area 40% excess of
surface area
thus RBCs become 2.5x more stretchable (compared
to resting diameter)
membrane is 100x more elastic than comparable
latex membrane, tensile strength is greater than that
of steel
depends on:
a. geometry
b. cytoplasmic (Hb) viscosity: Hb = viscosity
o > 36% Hb compromises RBC deformability
and shortens RBC life span
o RBCs age surface area decrease, HB
retained Hb concentration rises RBCs
unable to pass through splenic pores RBCs
consumed by splenic macrophages
c. elasticity/pliancy

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RBC MEMBRANE LIPIDS

8% CHO, 52% proteins, 40% lipids
RBC has a bilayer of cholesterol and phospholipids in
equal proportion
cholesterol and lipids may also redistribute laterally

A. Phospholipids
impenetrable fluid barrier
hydrophilic polar head groups oriented
toward both the aqueous plasma and the
cytoplasm
hydrophobic nonpolar acyl tails central layer
sequestered from aq plasma and cytoplasm
maintain gradient
reseal rapidly when membrane is torn
asymmetrically distributed
outer: phosphatidylcholine and sphingomyelin
inner: phosphatidylserine and
phosphatidylethanolamine
distribution is energy dependent, relies on
flippases, floppases and scramblases

Phosphatidylserine
only negatively charged
flips to the outer layer towards senescence

B. Cholesterol
esterified, hydrophobic
parallel to phospholipid acyl tails
one molecule per phospholipid molecule
hydrophilic portion: -hydroxyl group
tensile strength
limits redistribution when increased
reduced when bile salt decreases

Acanthocytosis
membrane loses strength due to deficiencies in
enzymes that maintain cholesterol concentration

Glycolipids
sugar-bearing lipids
5% of external half of RBC membrane
associate in clumps or rafts
support CHO chains to help form glycocalyx

Glycocalyx
negatively charged
prevents microbial attack
protects RBC from mechanical damage caused by
adhesion to neighboring RBCs or to the
endothelium
bear a few copies of carbohydrate-based blood
group antigens (e.g. ABH and Lewis)

**********
RBC MEMBRANE PROTEINS

A. Transmembrane proteins
support surface CHOs by glycosylation
transport and adhesion sites
signaling receptors
disruption leads to changes in osmotic tension of
cytoplasm
disruption in adhesion proteins adhesion then
fragmentation or vesiculation loss of
deformability senescence
signal transduction: activation of
submembranous G proteins initiating cellular
activities
complexes + anchorages = structural integrity
w/in proteins = ability to retain biconcave
shape despite deformability
vertical membrane structure
support carbohydrate-defined blood group
antigens
Band 3 and Glut-1
support ABH system carbohydrate
determinants
provide peptide epitopes
Glycophorin A
carries M and N determinants
Glycophorin B
carries Ss determinants

RH system
employs two multipass transmembranous
lipoproteins (D and CcEe, respectively, but requires
RhAG) and a multipass glycoprotein that each
cross the membrane 12 times

Phosphatidylinostol
anchors for:
decay-accelerating factor (DAF/CD55)
membrane inhibitor of reactive lysis (MIRL.CD59)
the two link to PI via a glycan core

Paroxysmal nocturnal hemoglobinuria
deficiency in DAF and MIRL
susceptible to complement-mediated hemolysis
PIG-A requires mutation

Sodium dodecyl sulfate-polyacrylamide gel
electrophoresis
SDS-PAGE
bands identified w/ Coomassie blue dye
glycophorins are stained using periodic-acid Schiff
dye (PAS)

Adducin:band 3; dematin:glut-1


B. Skeletal proteins
1. -spectrin and -spectrin
form antiparallel heterodimer held together by
a series of lateral bonds
form hexagonal lattice
provides lateral membrane stability
peripheral proteins
triple helical repeats of 106 amino acids each
= 20 repeats; = 16 repeats

Actin and protein 4.1
join ends of - heterodimers

Actin
form short filaments of 14 to 16 monomers whose
length is regulated by tropomyosin

Adducin and tropomodulin
cap the ends of actin

Dematin
stabilize the actin

Vesicles
blebs, lipid membrane peels off

Hereditary ellipsocytosis
ovalocytosis
arises from one of several autosomal dominant
mutations affecting spectrin dimer-dimer lateral
bonds or the spectrin-ankyrin-protein 4.1 junction
membrane fails to rebound from deformation
**********
OSMOTIC BALANCE AND PERMEABILITY

RBC membrane is impermeable to Na+, K+ and
Ca2+, water HCO3
-
and Cl
-


Aquaporin-1
transmembranous
forms pores/channels for inward flow of water

ATPase-dependent/Energy-dependent cation pumps
maintain 1:12 Na
+
and 25:1 K
+


Colloid osmotic hemolysis
cell swells, becomes spheroid, cell ruptures, spills Hb

Ca
2+
ATPase
extrudes calcium
maintain low intracellular levels of calcium
controlled by calmodulin