Vol.2, No.3, 220-227 (2010) Natural Science
http://dx.doi.org/10.4236/ns.2010.23034
Protein phase instability developed in plasma of sick
patients: clinical observations and model experiments
Tatiana Yakhno
Department of Radio-Physical Methods in Medicine, Institute of Applied Physics RAS, Nizhny Novgorod, Russia;
Received 6 November 2009; revised 1 January 2010; accepted 26 January 2010.
ABSTRACT
This article discusses the causes of formation
of micron-size protein structures in liquid plasma
or serum of the patients with different diseases,
which are accompanied by inflammatory reac-
tions. Self-organizing processes in sessile dry-
ing drops of natural and model biological liq-
uids are used for study of possible mechanisms
of development the protein phase instability in
serum. There was shown that violation of opti-
mal ratio between albumin and osmotic active
components could lead to loss of albumin ag-
gregative stability and albumin coagulation
structures formation. Possible role of these
structures in pathogenesis of inflammation is
discussed.
Keywords: Protein Aggregation in Serum;
Mechanisms of Inflammation
1. INTRODUCTION
In 2003 we reported that micron-size protein structures
were observed in liquid samples of serum and plasma of
patients with different diseases in acute stage [1]. We
could not give a correct explanation of this phenomenon
at that time, but it seemed to be very interesting and im-
portant for blood physiology as one of possible nonspe-
cific reactions of blood proteins accompanying inflam-
mation. When these samples of plasma or serum were
placed in the form of drops on glass for drying, the pro-
tein structures usually partly melted, and formed a
structured film consisting of agglutinated particles. In
serum and plasma of healthy people such structures ap-
peared only at a certain stage of drying, forming a
light-diffusing ring around the central part of the dried
drops [2]. Thus, we suggested that appropriate condi-
tions for protein structure formation in normal biological
fluids were developed at a definite moment during drop
drying. We believe that these conditions may be found in
model experiments using protein-salt water solutions.
Recently, protein phase transitions are widely studied in
the bulk of model solutions by means of static and dy-
namic light scattering, small-angle neutron and x-ray
scattering [3-8]. Phase separation is achieved by adding
precipitation agents: inorganic salts [4,5,8], polyelectro-
lytes and organic solvents [3,4,8], as well as by changing
the concentration, temperature, and pH level [4,5,7,9].
Experimental studies show that protein precipitation by
salts requires an electrolyte concentration in the range of
1-10 mole [9]. Protein aggregation in cells and biological
fluids is also a subject for investigation from the point of
view of crowding, because biochemical processes proceed
in a medium with high concentration (50-400 mg/l) of
macromolecules of different types [10-12]. This may lead
to formation of nonfunctional protein aggregates, which
upset balance between the active components, corrupting
their functions [12] and may even be toxic [13].
The critical protein charge required to induce pro-
tein-polyelectrolyte complex formation between bovine
serum albumin (BSA) and some synthetic polyelectro-
lytes was found to vary linearly as a function of square
root of ionic strength [14]. Formation of intrapolymer
complexes between human serum albumin (HSA) and
polyethylene glycol (PEG) showed formation of a wa-
ter-soluble complex the size of which varied depending
on both, ionic strength and molecular weight of PEG but
remained unaltered when the mixing ratio of PEG and
HSA was varied [15]. Serum glycosaminoglycans, such
as heparin, chondroitin and glucuronic acid, are native
polyelectrolytes [16], so it is reasonable to expect their
complex formation with HSA under certain conditions. It
is well known that abnormal protein aggregation is re-
sponsible for a variety of serious diseases, including eye
cataracts [17-19], sickle-cell anemia [20] and Alzheimer
disease [21]. It was shown also that the dynamics of
phase transitions in sessile drying drops of biological
liquids can be used as a sensitive parameter for medical
diagnostics [22-24].
The goal of this work is to attract the attention of
medical blood researchers to the phenomenon of protein
structure formation in native human biological fluids and

T. Yakhno / Natural Science 2 (2010) 220-227
221
to display factors that may be responsible for this phe-
nomenon using the drop drying model. Here we show
the data of model experiments with protein-salt water
solutions, and compare them with the data of our clinical
investigations carried out in 2000-2003.
2. MATERIALS AND METHODS
We used bovine serum albumin solutions (BSA, 68 kDa,
Sigma, USA) in distilled water or in salt-water solutions
with different protein/salt ratios. Salts (NaCl, KCl) were
labeled “chemically pure” (“Reaktiv, Inc.”, Russia). Ex-
periments were also performed with lyophilized prepara-
tions of human serum proteins supplied by OOO IMTEK,
Russia: 1) Human serum albumin (HSA, Sigma, USA,
#A-1653, 67 kDa, pI ≈ 4.7, E (280 nm, 1%) = 6.7); 2)
immunoglobulin G (IgG, 150 kDa, E (280 nm, 1%) = 14);
3) immunoglobulin M (IgM, 900 kDa, pI ≈ 7.2, E (280
nm, 1%) = 12); and 4) fibronectin (Fn, 420 kDa, E (280
nm, 1%) = 13). Also 10% wt food gelatin solution in
distilled water or in physiological salt solution was used.
To clarify a contribution of macromolecular serum
components to albumin aggregation, model protein mix
was prepared (Table 1). The mixed sample was com-
pared with pure HSA-salt water solution of the same
concentration. All solutions were prepared without buff-
ering, a day prior to experimentation, refrigerated over-
night and allowed to come to room temperature before
testing. The samples under study were placed, using
micropipette, onto clean glasses in the form of drops 3 µl
in volume (6-8 drops for each sample), and let for drying
in room conditions. Morphological observations were
carried out during drying, and 2-3 days after placing on
the glasses, using MБС-10 and Люмам-И-3 microscopes
with video camera computer setup.
Samples of blood plasma and serum were obtained
from 30 clinically healthy donors (the material supplied
by Hemotransfusion Station, Nizhny Novgorod); 18 pa-
tients with viral hepatitis B and C in acute stage (the
material supplied by the Hepatological Center, Nizhny
Novgorod); 30 patients with burn disease, and 8 patients
with diseases of articulations of inflammatory and de-
generative character (supplied by the Federal Burn
Treatment Center, Nizhny Novgorod Research Institute
of Traumatology and Orthopedics); 40 women after nor-
mal or premature (second- and third-trimester) childbirth
(supplied by the maternity and child-welfare services of
Nizhny Novgorod); one patient with Waldenstrom’s
macroglobulinemia, and seven patients with parapro-
teinemic hemoblastosis, with genetically modified IgG
(supplied by the Research Institute of Epidemiology and
Microbiology, Nizhny Novgorod). The samples were
transported at + 4°C, heated up to room temperature,
and then analyzed. The test fluids were applied on
chemically clean glass slides either as small drops (six
to seven 5-ul drops per slide) or as large drops (0.5 ml
per slide). The slides were dried in room conditions (T
= 18 – 22° C, H = 60 – 70%) for 24-48 h. Thus, drying
of the fluids occurred under different thermodynamic
conditions depending on their volume and the shape of
the drops. Some of the samples (small droplets) were
dried under an МБС-10 microscope fitted with a televi-
sion camera connected to a computer, so that phase tran-
sitions in the fluid could be recorded. After drying, the
drops were studied in a Люмам-И3 or МБС-10 micro-
scopes under conventional illumination conditions. Bio-
chemical data of blood of the patients under investigation
were obtained from the corresponding clinical laborato-
ries, and statistical data were calculated using the Excel
program.
3. RESULTS
The dried drops of protein solutions had a disc-like
shape with a thick protein ring around the edge and a
thinner circular central zone. Salt-free solutions formed
homogenous transparent dried spots (Figure 1(a,b)),
while protein-salt water dried drops had distinct concen-
tric zones: external protein ring, ring of light-diffusing
protein structures, ring of clear gel, and central zone of
shrinking gel (Figure 1(c,d)) due to salt crystallization
in it [2]. The above-named protein structures became
visible mostly on the second day of drying, after partial
evaporation of film water. So, when a drop is watched
from the edge to the center in a transmitted light micro-
scope, a homogenous layer of protein film is visualized
first, then single micron-size protein aggregates are set-
tled on the protein layer, after that fractal complexes are
formed of these separated structures which finally turn
into gel (Figure 1(e,f)). This principal sequence is the
same for globular (BSA, HSA) and non-globular (gelatin)
protein-salt water solutions regardless of protein and salt
type and con centration. The difference is in morpho-
logical features and in the width of these zones, depend-
ing on the ionic strength/protein ratio. The protein ring in
Table 1. Content of model protein solutions in 0.9% NaCl water solution.
№ Sample content Total protein, g/l HSA, g/l Fn, g/l IgG, g/l IgM, g/l
1 HSA + Fn + IgG + IgM 84.5 70.0 0.3 12 2.2
2 HSA (control) 84.5 84.5 0.0 0.0 0.0

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222
dried drops moves to the edge when the salt/protein ratio
in the initial solution increases (Figure 2).
Earlier we found that IgG, IgM and Fn exert a consid-
erable effect on both, the morphological and dynamic
parameters of fluid structuring in drying drops through a
change in the surface tension and viscoelastic properties
of the adsorption layer at the fluid–air interface [25,26].
The goal of the current work is to elucidate how these
macromolecular serum components influence protein
structure formation during drop drying. We replaced part
of HSA in physiological salt solution by different serum
macromolecules with the same weight proportions (Table
1), and saw a different type of protein structures in dried
drops in comparison with the control sample (Figure 3).
They looked like large-scale agglutinated particles fol-
lowing the homogenous protein ring at the drop’s
edge. We chose the concentrations of the macromolecular
Figure 1. Dried drops of protein water solutions and their
fragments. (a) – 7% wt BSA in distilled water; (b) – 10%
wt gelatin in distilled water; (c) - – 7% wt BSA in 0.9% wt
NaCl solution; (d) - 10% wt gelatin in 0.9% wt NaCl solu-
tion; (e) – fragment of a peripheral zone of a dried drop C
with protein structures on the homogenous protein layer; (f)
– fragment of a peripheral zone of a dried drop d with
protein structures on a homogenous protein layer. Arrows
(c, d) show light-diffusion rings of protein structures. a-d
shown at × 28 original magnification using МБИ-3 mi-
croscope. (e) and (f) shown with originаl magnification x
140 (e) and 70 (f) using Люмам-И3 microscope and video-
camera computer setup.
Figure 2. Dried drops of NaCl/BSA water solutions with
different NaCl/BSA ratio (right), and their peripheral zones,
are labeled with rectangle (left). NaCl/BSA weight concen-
trations are the following: (a) – 1.3/6.0; (b) – 1.5/5.0; (c) –
1.8/4.3; (d) – 2.2/3.4. Increasing in salt/albumin ratio leads
to moving protein structures zone to the drop’s edge (left).
(a-d) (left) shown at × 28 original magnification using a mi-
croscope МБИ-3. (a-d) (right) shown at origin magnification
× 70 using a microscope Люмам-И3 and video-camera
computer setup.
components approximately close to their normal con-
centration in blood [27].
These observations suggest that protein structures be-
gin to form at a definite stage of drying of protein-salt
water solutions, and this phenomenon is inherent in all
the samples under study. Ionic strength, protein concen-
tration and serum macromolecule components present in
the initial solution influence structure formation. Sur-
prisingly, sometimes we could see micron-size protein
structures in liquid samples of plasma or serum of pa-
tients with different diseases (Figure 4(b-d)). They were
never observed in liquid samples of healthy people (Fig-
ure 4(a)), but were usually formed at a definite stage of
drop drying. Peripheral protein zones in dried drops of
blood serum of healthy patients had the same order as

T. Yakhno / Natural Science 2 (2010) 220-227
223
Figure 3. Influence of macromolecular serum components
on protein structure formation in drying drops. (a) – a dried
drop of mixed protein solution (HSA+Fn+IgG+IgM) in
0.9% wt NaCl water solution with total protein concentration
84.5% wt; (c) – fragment of peripheral zone of the drop a; (b)
– a dried drop of 84.5% wt HSA solution in 0.9% wt NaCl
water solution (control, see Table 1); (d) – fragment of pe-
ripheral zone of the drop b. In contrast to the control sample,
zone of protein structures in c looks as large-scale aggluti-
nated particles. a and b shown at × 28 original magnification
using МБИ-3 microscope. c and d shown at original magni-
fication × 70 using Люмам-И3 microscope and video-camera
computer setup.
Figure 4. Fluid sessile drops of biological liquids at the very
beginning stage of drying. (a) – serum of practically healthy
donor; (b) – serum of patient with chronic hepatitis b and
chronic hepatitis c; (c) – plasma of patient with burn disease; (d)
– serum of patient with coxarthrosis. Shown at original magni-
fication using a microscope МБИ-3. Drop diameter is 5 mm.
Figure 5. Peripheral zones of dried drops of serum. (a) –
practically healthy donor; (b) and (c) – two patients with
burn disease; (d) – patient with paraproteinemia (overpro-
duction of genetically modified IgG). The drop a has a ho-
mogenous protein film over the edge, then zone of protein
structures begins. In the drops b-d zone of homogenous pro-
tein is absent. This zone is represented by agglutinated pro-
tein particles. Shown at original magnification × 70 using
Люмам-И3 microscope and video-camera computer setup.
model protein-salt water solutions: homogenous protein,
ring of protein structures, and gel (Figure 5(a)). In the
serum of patients with burn disease and paraproteinemia,
the very first zone
was a structural protein film instead of a homogenous
one (Figure 5(b-d)). This film consisted of a lot of small
agglutinated protein structures. Microscopic investigation
of big (0.5 ml) drops showed that irrespective of the dis-
ease type, these protein structures didn’t differ from each
other in principle (Figure 6).
Albuminemia is a usual state of acute-phase reactions
of different diseases [28]. Thus, average content of total
protein and albumin was estimated by the example of
burn disease patients and healthy donors. Also, plasma
osmolarity was calculated using the empiric equation:
Osmolarity (mosm) = 195.1 + 0.74 Na (mM/l) + 0.25
N of blood urea (mg %) + 0.03 Glucose (mg %).
Figure 7(a-c) shows the results of the calculations.
Thus, burn disease was characterized by total proteine-
mia due to albuminemia and a tendency to excess of
plasma osmolarity. It is clear that the ratio of osmotic
active components to albumin content in plasma in-
creases in burn disease, and tends to norm during treat-
ment (Figure 7(d)). At the same time, the peripheral
zone of dried serum drops becomes more homogeneous.

T. Yakhno / Natural Science 2 (2010) 220-227
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Figure 6. Protein structures in dried samples of serum of
patients with different diseases. (a) and (b) – two patients
with acute viral hepatitis b; c and d – two patients with burn
disease; (e) – a patient with Waldenstrom’s macroglobuline-
mia; (f) – a woman after premature birth. Shown at original
magnification × 70 (a-d and f) and 140 (e) using Люмам-И3
microscope and video-camera computer setup.
4. DISCUSSIONS
Desiccated sessile drops of colloidal suspensions are
good objects for studying liquid instabilities and non-
equilibrium pattern formation [29]. The main principles
of deposit ring formation over the circle (“coffee drop
deposit”), as well as pattern formation, depending on
particle size, concentration, ionic strength, and surfactant
presence [30] were revealed in drying drops of model
colloidal suspensions of polystyrene microspheres in
water. Detailed description can also be found in [31,32].
The point is that a redistribution of protein content along
drop radius occurs at the beginning of drop drying: up to
70% of albumin moves to the drop’s edge and solidifies
there, forming a ring, whereas the central part of the
drop is still a liquid [2]. At the same time, salt concentra-
tion enhances in the liquid center due to water evapora-
tion, and the salt/protein ratio increases. It can lead
firstly to liquid-liquid protein separation in the form of
coalescention, and subsequent solidification of neogenic
micelles because of outside osmotic pressure increases.
AFM investigation of appropriate zone of a dried drop of
a protein-salt water solution allowed finding a structure
that seemed to be a precursor of a micron-size protein
aggregates that were observed via optical microscope. It
consisted of rounded substances–subunits with a radius
about 50 nm [2]. Then, those quasi-solid albumin parti-
cles began to aggregate into micron-size structures
forming fractal clusters, which finally transformed into
gel. Thus, a gradual increase of the salt/albumin ratio
was a driving force for this cascade of albumin struc-
tures formation in drying drops of albumin-salt water
solutions. In a similar manner, increasing of osmolar-
ity/albumin ratio in the samples of biological liquids was
one of the main promoters of structure formation too.
Increasing in concentration of macromolecules of dif-
ferent nature also promoted loss of colloid stability. The
capacity of large unfolded protein molecules to bind and
transform water to a structured state [33,34] decreases
the volume of free biologically active water, which plays
an important role in prevention of coagulation. On the
other hand, surfactants and polyelectrolytes under appro-
priate conditions can interplay with albumin, forming
coacervates.
Cooper, C.L., et al. [35] (Figure 4) succeeded in
viewing by eye in a tube coacervate/dilute equilibrium
phase separation and polyelectrolyte-protein complexes
formation (BSA, labeled with fluorescein isothiocynate,
and poly (diallyldimethylammonium chloride).
Figure 7. The data of serum protein content and osmolarity
in norm compared with patients with burn disease. (a) – Total
protein (g/l); (b) – Albumin (g/l); (c) – osmolarity (mosm/l); (d)
– the dynamics of osmolarity/albumin ratio during treatment
in a patient with burn disease. Microscopic images show the
peripheral zones of dried drops at the beginning of treatment
(left) and after two weeks of treatment (right). One can see
the transformation of a structural protein film to a homogenous
film.

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Coacervates were prepared at pH 9.5 and I = 0.1M NaCl.
It is interesting to compare this data with our observa-
tions of the same phenomenon in serum of a patient with
Waldenstrom’s macroglobulinemia [1], when serum in a
tube segregated spontaneously into two transparent liq-
uid fractions differing in light-refracting capacity: lower
density at the top and higher density at the bottom. Upon
drying, the structures of interest were observed in the
bottom fraction only, as well as in the thoroughly mixed
whole serum. The transparency of these complexes meant
that they were well hydrated, and “invisible”. During film
water evaporation they lost their water shell, and became
visible. Appearance of such structures in liquid samples
of biological fluids means that this complex system –
serum or plasma – transforms to a non-equilibrium state
due to the presence of blood macromolecules, including
acute-phase proteins, and osmotic active components
which violate mutual balance and lead to loss of colloid
stability.
Albumin is the most mysterious multifunctional blood
protein. In norm, albumin realizes ligand-binding and
transport functions, as well as supports colloid osmotic
pressure of plasma and regulates neutrophyl function [36].
It has already been found that albumin specifically binds
with low molecular weight molecules that might be im-
portant diagnostic and prognostic indicators of diseases
[37]. In clinics, albumin has been intensively used in
critical conditions, including vascular collaps in seri-
ously ill patients [38]. At the same time, it was shown
that highly purified commercial albumin preparations for
laboratory use may contain significant amounts of albu-
min polymers [39-41]. There was an increase in death
among patients who were treated with human albumin
solution for burns, hypoalbuminaemia and hypotension
[42] by up to 6%. Pro and contra for using albumin in
clinical practice are widely discussed in the literature
[43].
It was shown that albumin polymers [44] and their
glycation olygomers [45] promote erythrocyte rouleaux
formation in blood, whereas molecular protein prevents
erythrocyte aggregation [46]. Armstrong, J.K., et al.
(2004) [47] have found that dimension of hydrodynamic
radius of polymer or macromolecule is the main criterion
of its erythrocyte aggregation capacity: if it does not
exceed 4 nm, it prevents aggregation, and if it is more
than 4 nm, it promotes it. Thus, in accordance with these
data, molecular albumin is a promoter of erythrocyte
disaggregation, and albumin polymers (or albumin co-
agulation structures) are promoters of their aggregation.
Shacter, E., et al. (1993) [48]have reported that albumin
polymers presented in commercial samples of albumin
can stimulate interleukin-6 and prostaglandin E2 produc-
tion by macrophags in vitro and in vivo. The albumin
fraction with molecular weight ≥ 600 kD had the highest
stimulation activity. It means that this polymer contains
10 or more monomers. The authors [48] believe that this
is a new mechanism of cytokine activation that triggers a
cascade of inflammatory reactions in organism.
This short review demonstrates that protein coagula-
tion structures formed in biological fluids due to loss of
their colloid stability can start the same cascade of
pathophysiological reactions. In addition, protein coagu-
lation leads to decreasing of blood oncotic pressure and
to water penetration into intercellular space which en-
closes a vicious circle forming edema and worsening
microcirculation. Thus, this phenomenon requires fur-
ther investigation, strong laboratory control and elabora-
tion of methods for its physical-chemical corrections if
necessary. The drop drying process is a natural model of
“functional loading” on biological liquids by decreasing
albumin concentration and increasing ionic strength,
which allows one to evaluate the degree of its colloid
stability.
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