Engineering, 2012, 5, 176-179
doi:10.4236/eng.2012.410B046 Published Online October 2012 (http://www.SciRP.org/journal/eng)
Mathematical Modeling of Hemoglobin Release under
Hypotonic Conditions*
Ivana Pajic-Lijakovic1, Branko Bugarski1, Milenko Plavsic2
1Dept. of Chem. Eng. University of Belgrade, Faculty of Technology and Metallurgy, Belgra de, Serbia
2Dept. of Polym er E ng., University of Belgrade, Faculty of Technology and Metallurgy, Belgrade, Serbia
Received 2012
ABSTRACT
Mathematic al model i s develo ped to est imate hemoglo bin release und er hypoto nic condit ions at micro scopi c level. The phenomenon
of hemoglobin (Hb) release depends on: 1) the dynamics of repeated opening of hemolytic holes and 2) the radial fluctuations of lipid
membrane. Both processes are sensitive to the rate of ionic strength decrease within the surrounding medium. Influence of the rate of
ionic strength decrease on hemoglobin release is quantified by the model parameters: 1) the specific decrease of erythrocyte radius
and 2) the specific decrease of hole radius during single opening time period of hemolytic hole. The prediction of released amount of
Hb influenced by the conductive mechanism is equal to 2.9 %. The prediction of total released amount of Hb influenced by the con-
ductive and convective mechanisms is approximately equal to 4 % of the initial amount of Hb within erythrocyte.
Keywords: Hemoglobin Transport; Hemolytic Hole; Thermodynamics; Cellular Biology and Engineering; Mathematical Modeling
1. Introduction
In last decades, many research groups attempted to design op-
timal “large-scale” meth od s for isol ation and purification of Hb,
with an aim to obtain highly purified, structurally, and func-
tionally preserved molecule, which has been used as a starting
material in production of blood substitutes, food additives for
prevention of anemia, and/or standards for clinical diagnostics
of different molecular variants of Hb. Different procedures of
isolation and purification of Hb from mammals erythrocytes
exist [1-2]. Nevertheless, in many of them, first isolation step is
osmotic hemolysis. We have optimized osmotic hemolysis of
outdated human erythrocytes at macroscopic level ensuring
efficiently mixing pattern in bioreactor and regulating the rate
of ionic strength decrease within the surrounding solution as
reported by Bugarski and Dovezenski [2]. Rate of ionic strength
decrease affects rheological response of cell membranes de-
pendently on cell morphology. It influences the processes at
microscopic level: the erythrocyte swelling, the repeated os-
motic holes opening and on that way the intra-cellular hemog-
lobin transport and the hemoglobin release through hemolytic
holes. The erythrocyte swelling is induced by solvent in-flux
driven by total osmotic pressure difference between in-
tra-cellular region and external medium under hypotonic condi-
tions. After in-flux of solvent, in-flux of Na+ cations and
out-flux of K+ cations follows. The formation of a hemolytic
hole represents the result of membrane relaxation on swelling
caused by solvent in-flux [3-7]. Consequently, such relaxation
induces rearrangement o f tr ans-membr a ne prote i n ba nd 3 which
includes formation of clusters [6]. The hole is bordered by a
ring structure of band 3 molecules [4,6].
Deeper insight into phenomenon of osmotic hemolysis oc-
curred at microscop ic level i s necessar y step for further process
optimization. Several investigators have studied complex dy-
namics of reversible osmotic holes formation through many
open ing time cycles [ 3-7]. However, little is reported about the
mechanism of intra-cellular Hb transport depending on the
dynamics of holes opening. At the same time, some aspects of
the intra-cellular transport phenomena are important for appli-
cation s of erythro cytes as carriers for vario us active su bstan ces.
It is n ecessary to co nnect the rheo logical ch aracteri stics of lipid
membrane with hemoglobin intra-cellular transport. Main goal
of this investigation is the modeling of hemoglobin in-
tra-cellular transport and h emoglobin rel ease in order to predi ct
and optimize osmotic hemolysis at microscopic level based on
the proposed model parameters. Model is formulated based on:
(1) the local balance equations of Hb by including (2) the
changes of erythrocyte volume and surface described using
Young-Laplace equation [7] as well as (3) the changes of os-
motic ho le area describ ed statistically during the single opening
time period of osmotic hole in our previous work [6].
2. Theore tical Par t
2.1. Phenomenological Background of the Model
The phenomenon of Hb intra-cellular transport under hypotonic
conditions is considered. We will formulate the mathematical
model for prediction of: 1) the intra-cellular Hb profiles within
erythrocyte during the single opening time period of hemolytic
hole and 2) the released amount of Hb. Necessary data for
model development are: 1) the volume of alread y swollen eryt-
hrocyte decreases 6 % during the single opening time period of
hemolytic hole, 2) the released amount of Hb after three re-
peated opening time periods of hemolytic hole is about 17 %, 3)
the averaged time period in which hemolytic hole is opened is
*Ministry of Education and Scien ce of Serbia

I. PA JIC-LIJAKOVIC ET AL.
177
equal to ts=0.27 s from Zade-Oppen [8]. Oth er necessary data
for the model development are: 1) the initial averaged volume
of swollen erythrocyte [7,9], 2) the radius of reversible opened
hemolytic hole [4], 3) the initial intra-cellular concentration of
Hb after erythrocyte swelling [7], 4) Stoke’s radius of Hb mo-
lecule [ 10] , 5) t he ef fectiv e di ffus io n co efficient estimated fro m
[11,12]. Additional consideration of rheological behavior of Hb
intra-cellular solution from [13,14] is necessary for model de-
velopment.
Intra-cellular volume fraction of Hb after swelling and before
release is Φ=0.289. Diffusion coefficient of Hb for infinite
dilution is D0=6.4x10-11 m2/s as reported by Shuklar [10]. Ri-
veros -Moreno and Wittenberg [11] and Doster and Longeville
[12] reported that the effective diffusion coefficient Deff de-
creases with the increase of the Hb concentration as the result
of increase the hydrodynamic and potential interactions be-
tween Hb molecules. They reported that Deff/D0~0.1 for Hb
volume fraction Φ>0.40. For Φ=0.289, the corresponding value
of Deff is approximately equal to Deff=0.2D0 based on Doster
and Longeville [12] considerations.
The Hb intra-cellular transport phenomena include conduc-
tive and convective mechanisms. The Hb conductive transport
is driven by pressure difference of Hb between intra-cellular
region and external medium. However, the Hb convective in-
tra-cellular flow represents the consequence of lipid membrane
radial fluctuations caused by surface energy changes. It is in-
duced by erythrocyte volume decreases during the single open-
ing time period of hemolytic hole.
2.2. Model Dev elop ment
We formulate the model for considering of the Hb intra-cellular
transport through erythrocyte to the already opened reversible
hemolytic hole as well as H b rele as e thro ugh the reversib le hole,
under hypotonic conditions. The model could be used for pre-
diction of the intra-cellular transport of some active substances
entrapped within erythrocyte for application of cells as drug
carriers. The initial time t=0 represents the time when the re-
versible hemolytic hole is opened. We suppose that the initial
hol e diameter RH0 is much higher than the Stoke’s radius of Hb
molecule. The final time for modeling consideration is t=ts (the
time for which hemolytic ho le is closed ). It rep resents the equi-
librium time for the single relaxation process of the membrane.
Before the model development, it is necessary to estimate the
erythrocyte volume Ve(t). The initial volume of erythrocyte
after swelling under hypotonic conditions is Ve0=4/3πRe03
(where Re0 is the erythrocyte radius at t=0). The volume de-
creases during the time period as the result of Hb out-flow. It is
expressed as: Ve=4/3πRe(t)3. The changes of erythrocyte vo-
lume and surface during the time period for which hole is
opened is considered as the set of the equilibrium states and
described using Young-Laplace equat ion as repo rted by Delano
[7] as dAe(t)γ=dVe(t)(P in(t)-Pout(t)) (where Ae(t) is the erythro-
cyte surface,
γ
is the sur face tension of erythro cyte membrane).
The interior and exterior pressures are expressed using van’t
Hoff’s Law as: Pin(t)=RTφinCin(t) and Pout(t)=RTφoutCout(t)
(where R is the universal gas constant, T is temperature, φin and
φout are the averaged intracellular and external osmotic coeffi-
cients respectively; Cin(t) and Cout(t) are the intra-cellular and
external solute concentrations respectively). The radiu s of eryt-
hrocyte Re(t) is expressed as Re(t)=2γ/ΔP(t) (where the total
pressure difference is ΔP(t)=Pin(t)-Pout(t)). Intra-cellular solute
concentration for intact cell is equal to 289 mol/m3 [7]. After
swelling, such concentration decreases up to Cin(0)=138.8
mol/m3. During the single opening time period of hemolytic
hole, the erythrocyte volume decreases about 6 %. After three
repeated opening time periods averaged content of Hb is 83 %
of its init ial value as reported b y Zade-Oppen [8]. Accordingly,
the averaged value of the released Hb content should be less
than 10 % per single opening time period of hemolytic hole.
For such preliminary condition, the intra-cellular solute con-
centration should be Cin(t)=147.5 mol/m3. We suppose that the
volume of external solution is much higher than the volume of
single erythrocyte. It indicates that the external solute concen-
tration Cout(t)~const as well as Pout(Cout(t))~const. The total
pressure difference increases during the single opening time
period of hemolytic hole as the result of the intra-cellular solute
concentration Cin(t) increase. We simplified the differential
changes of the total pressure difference as the linear phenome-
non: ΔP(t)=ΔP(0)eκt (where the model parameter κ quantified
the sp ecific increase of the total pressure difference). The radius
of erythrocyte is formulated as: Re(t)=Re(0)e-κt. The model pa-
rameter κ (the specific decrease of erythrocyte radius during
single opening time period of hemolytic hole) is equal to
κ=1/tsln(Re(0)/Re(ts)). It depends on the rate of ionic strength
decrease within the surrounding medium.
We consider the one-dimensional axial Hb transport through
erythrocyte to the opened hemolytic hole along x-axe. The bal-
ance equation for the Hb transport is expressed as:
( )( )( )( )
2
2
,,
,,
ff
x feff
c xtc xt
Uxt cxtD
tx x
∂∂
∂
=−+

∂∂ ∂
(1)
where x is the axial coordinate such that x€[0,2Re(t)], t is time
such that t€[0,ts], cf(x,t) is the local Hb concentration in the
erythro cyte, D eff is th e effect ive di ffusi on co efficient an d Ux(x,t)
is the axial velocity. We suppose that the local radial Hb con-
centration is constant within the volumetric increments ΔV(x,t)
=Ax(x,t)dx (where Ax(x,t) is the local cross section in axial
direction). The Hb concentration changes along x-axis as the
result of Hb release. Axial velocity is formulated based on the
equation of continuity as:
( )( )
( )
( )
,
,,
,
r
xr
x
A xt
UxtUxtA xt = (2)
where Ur(x,t) is the radial velocity, equal to Ur(x,t)=∂h(x,t)/∂t,
h(x,t) is the local radial distance such that h(x,t)=2[Re(t)2-
(Re(t)-x)2], Ar(x,t) is the surface increment equal to Ar(x,t)=
h(x,t)πdx. In that way changes of h(x,t) induce membrane radial
fluctuat ion s. The local cr oss secti on in axial dir ection is equal
to Ax(x,t)=1/4h(x,t)2π. The molar Hb out-flow through the re-
versible hemolytic hole to the external hypotonic medium is
expressed as :
( )( )( )( )
( )
( )
2
,
,,
e
f
x feffH
x Rt
c xt nt Uxt cxtDAt
tx
=
 ∂ ∂= −

∂∂

(3)

I. PA JIC-LIJAKOVIC ET AL.
178
where n(t) is the number of Hb moles which escape through the
hole. The surface area of hemolyti c hole AH(t) decr eases dur ing
the time period t€[0,ts] (where t s is the time such that AH(ts)=0).
The shape of the osmotic hole is nearly circular. Changes of the
area of the hemolytic h ole have been formulated as [ 6]:
( )
0
H
t
HH
At Ae
λ −
=
(4)
where AH0 is the in itial su rface area of the reversi ble he molytic
hole expressed as AH0=RH02π (RH0 is the initial radius of hole)
while λH is the specific decrease of hole radius equal to
λH=1/τrH (where τrH is the relaxation time for process of hole
closing). The model parameter λH strongly depends on the rate
of ionic strength decrease within the surrounding medium. We
introduce the first approximation for the value of the relaxation
time of the hemolytic hole as τrH~ts/3 which corresponds to the
boundary condition AH(ts)=0 [6].
The system of modeling equations (1-4) are solved starting
from the initial and boundary conditions:
1. At t=0 for pH=7.2 Hb molecules are anions uniformly dis-
tributed within erythrocyte. The initial Hb concen tration cf(x,0)
is cf(x,0)=c0s (where c0s=3.5 mol/m3).
2. At t=ts erythrocyte volume is equal to Ve(ts)=0.94V e0s.
3. Results and Discussion
The modeling considerations should point to the influence of
the dynamics of repeated opening hemolytic hole and the r adial
fluctuations of lipid membrane on hemoglobin intra-cellular
transport and hemoglobin release through the model parameters:
(1) the specific decrease of erythrocyte radius and (2) the spe-
cific decrease of hole radius during single opening time period
of hemolytic hole. The model parameters quantify process sen-
sitivity to changes of the ionic strength within the surrounding
medium. For high experimental rate of ionic strength decrease
the model parameters tend to infinity while for low experi men-
tal rate o f ioni c strength decrease t he model parameters t end to
zero. For experimental data from Zade-Oppen [8] the specific
decrease of erythrocyte radius was equal to κ=0.10s-1 and the
specific decrease of hole radius was equal to λH=11.1 s-1. We
tried to connect such experimental conditions from Zade-Oppen
[8] with Hb intra-cellular transport and Hb release. The pre-
dicted profiles of Hb concentration during the single opening
time period of hemolytic hole was calculated using (1) based on
the exper imental dat a from [4 ,5,7-12]. The profiles were shown
in Figure 1. Such profile represented the total result of both:
the convective and conductive mechanisms of transport. For
deeper insight into the influence of various mechanisms on Hb
intra-cellular transport we calculated separately the prediction
of: (1) the amount of Hb released as the result of the conductive
mechanis m and (2 ) the tot al amou nt of Hb released as th e resu lt
of both, the convective and the conductive mechanisms. It was
shown in Figure 2.
The prediction of released amount of Hb influenced by the
conductive mechanism was equal to 2.9 %. The prediction of
released amount of Hb influenced by both mechanisms was
approximately equal to 4 % of the initial amount of Hb within
erythrocyte. Zade-Oppen [8] experimentally determined that
the averaged amount of Hb released after three jumps (three
repeated opening time periods of hemolytic hole) for single
Figure 1. T he model prediction of hemoglobin concentratio n prof i le
within erythrocyte obtained from (1) during the opening time pe-
riod of hemolytic hole.
Figure 2. The released amount of hemoglobin obtained from (4)
during the openi ng time period of hemolyti c hole.
erythrocyte was equal to 17 % of the initial intra-cellular
amount of Hb. The averaged velocity of single cell during jump
was equal to vc=6.93±6.442 μm/s [8]. The corresponding aver-
aged velocity as the result of the conductive transport Deff/Δx
was calculated using the proposed model. It was equal to ~9.1
μm/s. The corresponding averaged velocity for the convective
transport was equal to ~1.8μm/s.
4. Conclusions
The results of this study point to the influence of the rate of
ionic strength decrease within the surrounding medium on the
hemoglob in release at micro scopic level bas ed on the modeling
considerations. The developed model connects the phenomenon
of hemoglobin release with the dynamics of repeated opening
of hemolytic hole and the fluctuations of lipid membrane. The
key model parameters for process optimization are: (1) the
specific decrease of erythrocyte radius and (2) the specific de-
crease of hole radius during single opening time period of he-
molytic hole. Higher experimental rate of ionic strength de-

I. PA JIC-LIJAKOVIC ET AL.
179
crease causes undesirable osmotic rupture of cells. For such
conditions, the model parameters tend to infinite. On the other
side, low experimental rate of ionic strength decrease suppress
osmotic hemolysis. For such conditions, the values of model
parameters tend to zero. For experimental data from
Zade-Oppen [12] the specific decrease of erythrocyte radius is
equal to κ=0.10s-1 and the specific decrease of hole radius is
equal to λH=11.1 s-1. The correspon din g predi ction o f released
amount of Hb influenced by the conductive mechanism is equal
to 2.9 %. The prediction of total released amount of Hb influ-
enced by the conductive and convective mechanisms is ap-
proximately equal to 4 % of the initial amount of Hb within
erythrocyte. It is obtained for the initial diameter of the reversi-
ble hemolytic hole equal to 3 μm.
5. Acknowledgements
The support by grants (# III 46010 and # III46001) from the
Ministry of Education and Science, Republic of Serbia is
grateful ly acknowledged.
REFERENCES
[1] S.M. Christensen, F. Medina, R.W. Winslow, S.M Snell, A.
Zegna, M.A. Marini, “Preparation of human hemoglobin Ao
for possible use as a blood substitute” J. Biochem. Biophys. Me-
thods 1988;17, pp. 143-54, 1988.
[2] B . Bugarsk i, N. Dovezen ski, H emofarm Konc ern. Verf ahren zu r
Herstellung von Hemoglobin, Deutsches Patentamt DE
19707508, 2000.
[3] M.R. Leiber, T.L. Steck, “A description of the holes in human
erythrocyte membrane ghosts”, J. Biol. Chem. 257,
pp.11651-11659, 1982.
[4] Y. Sato, H. Ya makose, Y. Suzuki, “Partic ipatio n of Band 3 Pro-
tein in Hypotonic Hemolysis of Hyman Erythrocytes”, Biol.
Pharma c. B ul . 16(2), pp. 18 8-194, 1993.
[5] A. Pribush, D. Meyerstein, N. Meyerstein, “Kinetic of erythro-
cyte swelli ng and memb rane hole forma tion in hypot onic media ”,
Bioch em . Biophys. Acta 1558, pp. 119-132, 2002.
[6] I. Pajic-Lijakovic, V. Ilic, B. Bugarski, M. Plavsic,“The rear-
rangement of erythrocyte band 3 molecules and reversible os-
motic holes formation under hypotonic conditions”, Europ. Bio-
phys. J. 39(5), pp. 789-797, 2010.
[7] M. Delano, “Simple physical constraints in hemolysis”, J. Theor.
Biol. 175, pp. 517-524, 1995.
[8] A.M.M. Zade-Oppen, “Repetitive cell 'jump' during hypotonic
lyses of erythrocytes observed with simple flow chamber”, J.
Micros c. 1 92 , pp. 54-62, 1998.
[9] G.B. Nash, H.J. Meiselman, “Red Cell and Ghost Viscoelasticity,
Effects of Hemoglobin Concentration and In Vivo Aging”,
Biophys. J. 43, pp. 63-73, 1983.
[10] R. Shuklar, M. Balakrishnan, G.P. Agarwal, “Bovine serum
albumin-hemoglobin fractionation: significance of ultra filtration
system and feed solution characteristics”, Bioseparation 9, pp.
7-19, 2000.
[11] V. Riveros-Moreno, J.B. Wittenberg, “The Self-Diffusion Coef-
ficients of Myoglobin and Hemoglobin in Concentrated Solu-
tions”, J. Bio l . Che m . 2 47(3), pp. 8 95 -901, 1 97 2.
[12] W. Doster, S. Longeville, “Microscopic Diffusion and Hydrody-
namics of Hemoglobin in Red Blood Cells”, Biophys. J. 93, pp.
1360-1368, 2007.
[13] A.M. Gennario, A. Luquita, M. Rasia, “Comparison between
Internal Microviscosity of Low-Density Erythrocytes and the
Microviscosity of Hemoglobin Solutions: An Electron Paramag-
netic Resonanc e Study”, Biophys. J. 71, pp. 389-393, 1996.
[14] D.P. Ross, A.P. Minton, “Analysis of non-ideal behavior in
concentrated hemoglobin solution”, J. Molec. Biol. 112, pp.
437-452, 1977.