S T U D I E S IN I N T E R F A C E SCIENCE
Dynamic Surface Tensiometry in Medicine
STUDIES
IN I N T E R F A C E
SERIES D. M 6 b i u s
SCIENCE
EDITORS and R. M i l l e r
Vol. I Dynamics of Adsorption at Liquid Interfaces
Theory, Experiment, Application by S.S. Dukhin, G. Kretzschmar and R. Miller Vol. 2
An Introduction to Dynamics of Colloids by J.K.G. Dhont Vol. 3 Interfacial Tensiometry by A.I. Rusanov and V.A. Prokhorov Vol. 4 New Developments in Construction and Functions of Organic Thin Films edited by T. Kajiyama and M. Aizawa Vol. 5 Foam and Foam Films by D. Exerowa and P.M. Kruglyakov Vol. 6 Drops and Bubbles in Interfacial Research edited by D. M6bius and R. Miller Vol. 7 Proteins at Liquid Interfaces edited by D. M6bius and R. Miller Vol. 8
Dynamic Surface Tensiometry in Medicine by V.N. Kazakov, O.V. Sinyachenko, V.B. Fainerman, U. Pison and R. Miller
Dynamic Surface Tensiometry in Medicine VALERY N. K A Z A K O V and OLEG V. S I N Y A C H E N K O
Medical University, Donetsk, Ukraine VALENTIN B. FAINERMAN
Institute of Technical Ecology, Donetsk, Ukraine ULRICH PISON
Virchow Klinik, CharitY, Humboldt Universit~it, Berlin, Germany REINHARD MILLER
Max-Planck-lnstitut f~r Kolloid- und Grenzfi6chenforschung Berlin-Adlershof, Germany
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Preface The dynamic and equilibrium properties of thin layers between two fluids, e.g., adsorption, interface tension, viscosity and elasticity, are primarily determined by the composition of the respective interracial layers. This composition, in turn, can be essentially different from that characteristic for the bulk phases, if substances of special amphiphilic structure, so called surfactants, are present in one or both adjacent fluids. Human biological liquids contain numerous low- and high-molecular weight surfactants. The human organism contains
interfaces with enormous surfaces.
The physicochemical and
biochemical processes taking place at these interfaces are extremely important for the vital functions of the organism as a whole, and the interracial properties may reflect peculiarities of age and sex, health and disease. The present book is the first attempt to systematically present the results of dynamic and equilibrium surface tensions measurements of serum and urine samples that were obtained from healthy human of various sex and age (more than 150 volunteers), and to compare these results with measurements of biological liquids obtained from patients suffering from various diseases (more than 1300 patients) or withmeasurements of amniotic fluid obtained from women at various stages of pregnancy. The work of M. Polfinyi in 1911 is probably one of the earliest research of surface tension of human biological liquids (cerebrospinal liquid). O. Ktinzel in 1941 has published the first systematic surface tension studies of serum and r surface tension behaviour of blood, r expired r
liquid. Only recently, studies of and amniotic liquid, gastric juice, saliva,
air and other human biologic liquids have been published; the most recent
examples are Hrncir & Rosina (1997), Brydon et al. (1995), Efentakis & Dressman (1998), Joura et al. (1995), Fell & Mohammad (1995), Adamczyk et al. (1997), Boda et al. (1997) and Manalo et al. (1996). However, such studies are still incomprehensive, and the methods used are often not reliable enough. One branch of medical science, pulmonary medicine especially
vi neonatology, has systematically used interfacial tensiometry for studying pulmonary surfactant. In this particular area, significant progress was achieved in the treatment of diseases related to alterations of the lung surfactant system (Pulmonary Surfactant: From Molecular Biology to Clinical Practice 1992, Surfactant Therapy for Lung Disease 1995, Pison et al. 1996). We believe that, similar to the progress in pulmonary medicine attributed to surface chemical studies of lung surfactant, progress in other medical branches could be expected through studies of interfacial characteristics of other human biological liquids. Such studies, however, will be successful only by using standardised techniques of dynamic and equilibrium surface tension measurements. For several years the authors of this book have been engaged in studies aimed at the improvement of the maximum bubble pressure method (Fainerman & Miller 1998a). These studies resulted in the development of the computer controlled tensiometers MPT 1 and MPT 2, commercially manufactured by LAUDA (Lauda, Germany), since 1993 and widely used in various areas of science and technology. The device is capable of measuring dynamic surface tensions within a wide range of surface lifetime (1 ms to 100 s for the standard version of the MPT 2 tensiometer). Therefore the initial intention was to apply just this apparatus to studies of dynamic and equilibrium surfacetymsion of biological liquids. However, already at the primary stage of the investigations it became apparent that the properties of serum and other biological liquids are essentially different from those characteristic to solutions of lowmolecular weight surfactants, for which the tensiometer MPT 1/2 were initially developed. Therefore, to achieve a sufficient reproducibility of the results, additional studies of the effect produced by the geometrical shape and surface properties of the capillaries had become inevitable. The results demonstrated that the capillaries for measurements of biological liquids should be essentially different from the standard developed for surfactants. The main theoretical and experimental issues related to the maximum bubble pressure technique as applied to biological liquids are presented in Chapter 2. We believe that one of the most significant achievements in this respect is the possible modification of the commercially
vii available instrument MPT 2, so that physicians, either practical or engaged in fundamental researches, can obtain relevant data and compare them with the values presented in this book. Therefore, the tensiometric parameters of biologic liquids taken from both healthy persons and patients suffering from various diseases, and the correlations between tensiometric and other data, i.e. clinical or biochemical parameters reported here, can be easily used, verified and extended by other scientists. A single example may illustrate how important selecting the right method is for studying of biological liquids. In the studies performed by Hrncir & Rosina (1997), the drop volume method was used to measure the surface tension of healthy persons' blood. The number of volunteers (adult males and females) was similar to that employed in our studies as reported in Chapter 3. It should be noted here that the drop volume method, as simple as it is on a first glance, is in fact rather complicated. This is due to the static and dynamic corrections to be used, and the fact that the viscosity of the liquid and the adhesive properties and geometric shape of the capillaries significantly affect the results (Miller & Fainerman 1998). For this method, only modern automated devices, e.g., TVT 1 (also manufactured by LAUDA, Germany) are capable of producing results, which can be compared with data obtained by other methods. Moreover, the application of this method for studies of surface tension of protein solutions (note that proteins are the main surface active constituents of blood) shows that in addition to significant scattering the results differ essentially from data measured by the Wilhelmy plate method (Tornberg & Lundh 1981). The average surface tension measured by Hrncir & Rosina (1997) for all samples of blood was 56 mN/m, which is comparable to the equilibrium surface tension of serum reported in Chapter 3. However, the standard deviation of data obtained in the drop volume study by Hrncir & Rosina (1997) was 19 raN/m, whereas the standard deviation of data obtained by the maximum bubble pressure method in Chapter 3 was 4 mN/m. This is significantly better. A more detailed discussion of the differences of the various methods in use for measuring dynamic surface tension of biological fluids will be provided in Chapter 3.
viii An important distinctive feature of the present experimental studies is that complete dynamic tensiograms of biological liquids have been measured. This makes the tensiometric studies more comprehensive and informative. Here again a single example will be given. In the study performed by Brydon et al. (1995) the equilibrium surface tension of cerebrospinal liquid was measured using the Wilhelmy plate method. Similarly to a number of earlier studies (Ktinzel 1936, 1941), the equilibrium surface tension values were found in the interval 59-64 mN/m. These results agree perfectly well with our data for equilibrium surface tension in the control group of patients
(60.4mN/m,
see Chapter7).
Moreover,
with
increasing protein
concentration, which is observed during some diseases, our studies support a trend revealed by earlier investigations that equilibrium surface tension decreases. While the Wilhelmy plate method provides only a single value for each liquid studied at a time, the dynamic tensiometry gives equilibrium surface tension, the shape of the tensiogram, characteristic slopes in particular intervals, and surface tension values at definite surface lifetimes. These dynamic characteristics are much more informative and exhibit better correlation with the pathology studied and the composition of the liquid, than an equilibrium surface tension value alone does. In addition to the measurement techniques, a correct interpretation and analysis of the tensiometric data obtained is extremely important. The kinetic theory of adsorption from solutions, and the theory of equilibrium adsorption layers of surfactant/protein mixtures (Miller et al. 1994, Dukhin et al. 1995, Fainerman & Miller 1998b) provide the basis for both the choice of the most characteristic parameters of tensiograms and the analysis of the results. Some theoretical models describing the adsorption of proteins are presented in Chapter 1. Chapters 4 to 8 will summarise dynamic surface tension data measured in biological samples that were obtained from patients with various diseases. Chapter 4 will give data from patients with kidney disease, chapter 5 from patients with rheumatic diseases, chapter 6 with pulmonary diseases, chapter 7 with diseases of the central nervous system, and chapter 8 with neoplasms. The authors of this book are indebted to a number of colleagues from the Donetsk Medical University (Donetsk, Ukraine), the Institute of Colloid Chemistry and Chemistry of Water
ix (Ukrainian National Academy of Sciences, Kiev, Ukraine), the Institute of Technical Ecology (Donetsk, Ukraine), the Max-Planck-Institut ftir Kolloid- und Grenzfl~ichenforschung (Golm, Germany), the University of Antwerp (Belgium), and LAUDA Dr. R. Wobser GmbH & CO. KG. (Lauda-KOnigshofen, Germany). We express our thanks to everyone who contributed to the development of experimental methods for performing these studies, the evaluation of theoretical models for discussing the experiments, and finally the preparation of the manuscript. A great tribute is given to the late Professor Paul Joos, one of the most distinguished scientists in the area of physical chemistry of surface phenomena. Professor Joos gave active support at the very beginning of our studies and he was among the authors of our first joint publication devoted to the tensiometry of human biological liquids (Kazakov et al.,
1995). The
comprehensive theoretical analysis of the hydrodynamic problems related to the maximum bubble pressure method, performed by Prof. S.S. Dukhin and Dr. V.I. Koval'chuk, showed us how to adjust the parameters of the MPT 1/2 tensiometers to modify this device for reliable measurements of biological liquids. We express our special thanks also to Ms. S.V. Lylyk for her technical assistance in performing the tensiometric studies. Invaluable help during the preparation of the manuscript was rendered by Dr. E.V. Aksenenko and D.V. Trukhin. The authors understand the deficiencies that are associated with the proposed publication. Sampling periods of biological liquids during the course of some of the diseases studied were quite narrow. In the discussion of revealed trends, we were unable sometimes to explain new results unambiguously in the framework of known mechanisms. Nevertheless, we hope that the readers will pay less attention to these drawbacks but rather keeping in mind the pioneering character of this book. The authors believe that dynamic interface tensiometry of human biological liquids is a fascinating new method and deserves a broad use for prospective studies of various diseases.
The authors
Donetsk/Berlin, 1999
References
Adamczyk, E., Amebrant, T., Glantz, P.O., Acta Odontologica Scandinavica, 55(1997)384. Boda, D., Eck, E., Boda, K., J. Perinat. Med., 25(1997)146. Brydon, H.L., Hayward, R., Harkness, W., Bayston, R., British J. Neurosurgery, 9(1995)645. Dukhin, S.S., Kretzschmar, G., Miller, R. Dynamic of Adsorption at Liquid Interfaces. Theory, Experiments, Application, in "Studies in Interface Science". Vol. 1, Elsevier, 1995 Efentakis, M., Dressman, J.B., European J. Drug Metabolism and Pharmacokinetics, 23(1998)97 Fainerman, V.B., Miller, R., In "Drops and Bubbles in Interfacial Research", in "Studies of Interface Science", D. MObius and R. Miller (Eds.), Vol. 6, Elsevier, Amsterdam, 1998a, p. 279-326 Fainerman, V.B., Miller, R., In "Proteins at Liquid Interfaces", in "Studies of Interface Science", D. M6bius and R. Miller (Eds.), Vol. 7, Elsevier, Amsterdam, 1998b, p. 51-102 Fell, J.T., Mohammad, H.A.H., International Journal of Pharmaceutics. 125(1995)327. Hmcir, E., Rosina, J., Physiological Research. 46(1997)319. Joura, E.A., Kainz, C., Joura, E.M., Bohm, R., Gruber, W., Gitsch, G., Zeitschrift ftir Geburtshilfe und Neonatologie. 199(1995)78. Kazakov, V.N., Fainerman, V.B., Sinyachenko, O.V., Miller, R., Joos, P., Lylyk, S.V., Ayko, A.E., Trukhin, D.V., Ermolayeva, M.N., Arch. Clin. Exp. Med. (Ukraine), 5(1995)3. Kazakov, V.N., Sinyachenko, O.V., Trukhin D.V., Pison, U., Colloids Surfaces A, 1998. Ktinzel, O., Deut. Zeitsch. Nervenheilkunde, 139(1936)265. Ktinzel, O., Ergeb. Inneren Med. Kinderheil, 60(1941)565. Manalo, E., Merritt, T.A., Kheiter, A., Amirkhanian, J., Cochrane, C., Pediatr. Res., 39(1996)947. Miller, R., Fainerman, V.B., In "Drops and Bubbles in Interfacial Research", in "Studies of Interface Science", D. M6bius and R. Miller (Eds.), Vol. 6, Elsevier, Amsterdam, 1998, p. 139-186 Miller, R., Joos, P., Fainerman, V.B., Adv. Colloid Interface Sci., 49(1994)249. Pison, U., Herold, R., Schtirch, S., Colloid Surfaces A, 114(1996)165. Pol~inyi, M., Biochem. Zeitsch., 34(1911)205. Pulmonary Surfactant: From Molecular Biology to Clinical Practice, Eds. B. Robertson, L.M.G. Van Golde and J.J. Batenburg, Elsevier, Amsterdam, 1992. Surfactant Therapy for Lung Disease, Eds. B. Robertson and H.W. T~iusch, Marcel Dekker Inc., New York, 1995. Tomberg, E., Lundh, G., J. Colloid Interface Sci., 79(1981)76.
Contents
xi
Preface
Chapter 1 - Theory of protein adsorption and model experiments
1.1.
Thermodynamics of protein adsorption at the liquid/fluid interfaces
1.2.
Adsorption kinetics
16
1.3.
Experimental studies of model biological liquids
20
1.4.
Influence of additives
26
1.5.
Summary
36
1.6.
References
37
Chapter 2 - Experimental technique and analysis of tensiograms
41
2.1.
Experimental methods
41
2.2.
The design of maximum bubble pressure tensiometer
43
2.3.
Theory of the maximum bubble pressure method
45
2.4.
Experimental technique
55
2.5.
Analysis of tensiograms
59
2.6.
Summary
64
2.7.
References
64
Chapter 3 - Dynamic interfacial tensiometry of biological liquids for healthy
68
persons
3.1.
Dependence of dynamic surface tension on sex and age of patients
68
3.2.
Dynamic surface tension of serum and amniotic liquid for pregnant women
81
3.3.
Summary
96
3.4.
References
96
Chapter 4 - Application of Surface Tensiometry in Nephrology
99
4.1.
Glomerulonephrites
100
4.1.1
Variation in surface tensiometric parameters for various forms of the disease
100
xii 4.1.2. Influence of particular serum and urine components on dynamic surface
118
tension 4.1.3. Effect of treatment on variations in surface tensiometric parameters
142
4.2.
Primary pyelonephritis and urolithiasis
152
4.3.
Diabetic nephropathy
163
4.4.
Other renal diseases
175
4.5.
Summary
183
4.6.
References
183
Chapter 5 - Surface tensiometry in rheumatology
191
5.1.
Pathogenesis of rheumatic diseases
191
5.2.
Systemic lupus erythematosus
195
5.3.
Rheumatism
207
5.4.
Sclerodermia systematica
215
5.5.
Rheumatoid arthritis
216
5.6.
Reiter's disease
228
5.7.
Psoriasis
229
5.8.
Gout
231
5.9.
Osteoarthrosis
236
5.10.
Effect of glucocorticoid therapy and plasmapheresis
237
5.11.
Summary
241
5.12.
References
241
Chapter 6 - Surface tensiometry in pulmonology
245
6.1.
Pathogenesis of respiratory diseases
245
6.2.
Bronchitis
258
6.3.
Bronchial asthma and other pulmonary diseases
264
6.4.
Dust pathology of respiratory organs
269
6.5.
Summary
281
xiii
6.6.
References
Chapter 7 - Surface tensiometry in neurology
282 286
7.1.
Tensiogram parameters for diseases of the nervous system
286
7.2.
Influence of the nosological form of an infection disease
297
7.3.
Role of patients age and duration of a disease
298
7.4.
Correlation between surface tension parameters and amount of proteins and
303
other components 7.5.
Role of tensiometry in therapy, diagnosis and prognosis
313
7.6.
Summary
321
7.7.
References
322
Chapter 8 - Interfacial tensiometry in oncology
324
8.1.
Pathogenesis of oncological disease
324
8.2.
Serum tensiograms for different tumour localisations
328
8.3.
Correlation between surface tensions and biological liquid's composition
335
8.4.
Influence of ~,-therapy on dynamic surface tensions
347
8.5.
Effects of operative treatments
353
8.6.
Summary
355
8.7.
References
358
List Of symbols
359
Subject Index
365
0
10.
This Page Intentionally Left Blank
Chapter 1
Theory of protein adsorption and model experiments In order to understand key parameters under discussion in this book, the dynamic surface tension characteristics of biological liquids, it is suitable to give a short introduction into the physical processes of adsorption of molecules like proteins and short-chain surface active molecules at liquid interfaces. This survey allows then to understand the role the dynamic surface tension characteristics can play in the analysis of correlations between these values related to the adsorption of all surface active component and medical findings related to particular diseases. The thermodynamics as well as the dynamics and mechanics of adsorption layers formed at liquid interfaces will be presented and discussed on the basis of up-to-date theoretical models.
1.1. Thermodynamics of protein adsorption at the liquid/fluid interfaces Human biologic liquids contain various surfactants capable of adsorbing at liquid interfaces and changing the surface (interfacial) tension. Adsorption processes involve proteins, phospholipids, and low molecular weight surfactants, which play a significant role in vital functions of the human organism, in respiratory processes and haematogenesis. The practical importance of the adsorption process of surfactants and polyelectrolytes, and in particular, proteins at liquid interfaces has stimulated the development of various theoretical models to describe the equilibrium and dynamic behaviour of this process. In most cases the adsorbed monolayers of surfactants, proteins and lipids exhibit non-ideal behaviour. To account for the non-ideality of surfactant monolayers in the equation of state and adsorption isotherm, the regular solution theory is generally used (Lucassen-Reynders 1966, 1972, 1982). Recently new theoretical models have been proposed considering actual physical phenomena within surfactant monolayers, in particular, the reorientation of adsorbed molecules (Fainerman et al. 1997), and the formation of dimers, trimers and larger clusters (Fainerman & Miller 1996a). The abundant surface active component in human blood is human serum albumin (HSA). Its concentration in the serum is 35 to 50 g/1. The properties of protein adsorption layers differ in a
2 number of aspects from those characteristic to surfactant monolayers. With protein adsorption surface denaturation takes place, leading to the unfolding of protein molecules within the surface layer, at least at low surface pressures. The partial molar surface area for proteins, in contrast to surfactants, is large and variable. This property, and also the large number of configurations possible for an adsorbed protein molecule, significantly exceeding that in the bulk, leads to an increased non-ideality in the surface layer entropy. This makes it impossible to apply the most simple models (Henry, Langmuir) for the description of protein adsorption layers. Various thermodynamic models for the protein adsorption at liquid interfaces were proposed. The interrelation between protein denaturation processes at the surface and the activity of the solvent (water) molecules was shown to exist by Ter-Minassian-Saraga (1981), while Joos (1975) had shown that the degree of surface denaturation decreases with increasing surface pressure. Lucassen-Reynders (1994) had analysed the effect of the size of mixed molecules on the entropy of protein surface layers. Joos & Serrien (1991) were the first to derive an equation for the adsorption of proteins possessing two modifications with different partial molar area. From this relation it follows that the surface pressure controls both the composition and the thickness of a protein surface layer. In particular, the part of molecules possessing the minimal surface area increases with increasing surface pressure FI. The concept proposed by Joos & Serrien (1991) was further developed for an arbitrary but discrete number of different configurations of protein molecules within the surface layer. Fainerman et al. (1996a) and Makievski et al. (1998) derived equations of state for the surface layer and isotherms of protein adsorption at liquid/fluid interface. These new relationships reflect the main feature of high molecular electrolytes possessing flexible chains: the capability of changing the partial molar surface area in response to a variation in surface pressure. The new equations describe the case of a non-ideal surface layer, that is, the non-ideality of enthalpy and entropy of mixing resulting from the differences in size of protein and solvent molecules. The effect of the electric charge of a protein molecule is considered, and contribute significantly to the surface pressure. The model of multiple discrete states of protein molecules within the surface layer was even generalised to the case of an infinite number of infinitesimal states (the continuum model). Recently the adsorption behaviour of concentrated protein solutions was considered (Fainerman & Miller 1998b).
Surface pressure and adsorption isotherms for proteins at a liquid/fluid interface can be derived from Butler's (1932) equation for the chemical potential ~t~ of ith state of a protein molecule within the surface layer: lt.t~ = bt~s + RTlnf.Sx~
--0"(1) i
(1.1)
and the corresponding equation for the chemical potential g~ within the solution bulk, g~ = l.ti~ + RTlnfi~x~
(1.2)
where g0s and g ~ are the standard chemical potentials, R is the gas constant, T is the absolute temperature, o is the surface tension, o3i are the partial molar areas, fi are the activity coefficient, x i =
N i /2N
i are the molar fractions, and
Ni are
the number of moles of the
i th
state. Here the superscripts 's' and 'a' refer to the surface (interface) and the bulk. For ideal bulk phases it follows from Eqs. (1.1) and (1.2) that
1 - I = - ~R| T [I I ( 1- ki_>~l0 i) +Inf~
(1.3)
0)0
Kic=(1 0i/n
(1.4)
where FI = o0- o is the surface pressure, c0 is the surface tension of the solvent (i = 0), 0 i = Fio3i , Fi are the adsorptions of component i, 00 = 1- ~-'~0i , ni = coi/o30, c is the total protein i>__l
bulk concentration. The coefficients K i = (x~/X~)xr__,0 for i_> 1 are the distribution coefficients of states at infinite dilution. It can be assumed that the value of o30 is close to the area of a water molecule, and therefore the adsorption of a protein molecule in the ith state leads to the desorption of ni = o3i/o30 water molecules. This is however only true when the adsorption layer comprises of water molecules, thus the adsorption layer is about 0.3 nm thick. Real adsorption layers of proteins are much thicker. Moreover, their thickness increases with the protein adsorption. Thus, from the
theoretical point of view, the procedure employed by Douillard et al. (1994), where the real thickness of the protein layer was taken into account, seems to be more reliable. In this case the portion of water molecules within the surface increases, while the number of desorbed water molecules per protein molecule becomes significantly larger than coiAo0. Fainerman et al. (1996a) assumed that COo- co~, coincides with the choice of the dividing surface defined by Lucassen-Reynders (1966, 1972, 1982)
2.r~ - 1/~z
(1.5)
i=0
Here ma is the mean partial molar area of all states
This choice of the dividing surface ensures that for each state the relation 0i = Fizz holds. As mr is the same for all states, Fimr is the surface molar fraction of the respective adsorption state. Therefore the transformation of Eqs.(1.1) and (1.2) into Eqs.(1.3) and (1.4) with the introduction of 0i instead of x~ is a rigorous procedure. Another important advantage which follows from the choice of the dividing surface according to Eq. (1.5) and mr~ according to Eq. (1.6) is the fact that there is no contribution due to the non-ideality of entropy of mixing to the solvent activity coefficient. Finally, using the Lucassen-Reynders' dividing surface one can exclude the adsorption layer thickness from further consideration so that the actual number of water molecules displaced from the adsorption layer during the adsorption of protein molecules needs not to be accounted for. It is seen from Eq. (1.5) that for ~ F~ = 0 the Lucasseni=1
Reynders'
dividing
surface
is shifted towards the
solution bulk
by the
distance
zX= (m0/mr).dH2o as compared to the Gibbs' dividing surface for which F0=0. Here dH2o is the diameter of a water molecule. For a saturated monolayer however, these two areas coincide with one another. Note that for proteins (mr~~ m0) the value of A becomes negligibly small, and therefore for any adsorption the Lucassen-Reynders' dividing surface coincides with the Gibbs' dividing surface.
The activity coefficients in Eqs. (1.3) and (1.4) can be represented in a form which accounts for the enthalpy and entropy of mixing, denoted by superscripts H and E, respectively (Lucassen-Reynders 1994, Makievski et al. 1998), In fs = In fisH + In fisE, i > _0
(1.7)
lnfi sH = a(1 -Fxo~x) 2, i>_ 1
(1.8)
lnfi sE = 1-co~ ~~ Fj - 1 - ni,i >1
(1.9)
j>o
In f~H = aF~o~x, 2 2
(1.10)
lnf~ E = 1 - c o 0 ~ F j = 0 j__0
(1.11) n
Here a is the intermolecular interaction constant, and Fz = )--'~Fi . For simplicity it can be i=l
assumed that the non-ideality of enthalpy of the surface layer is independent of the state of molecules within the surface, and therefore depends only on the total adsorption. Proteins are polyelectrolytes, i.e., they contain ionised groups. At the isoelectric point both hydroxyl groups and amino groups possess equal degree of ionisation, and thus the whole molecule is electro-neutral. In strong acidic media the hydroxyl groups become neutral and the molecule acquires an excess of positive charges, while a neutralisation of the amino groups in strong alkaline media results in a negative net charge of the protein molecule. Therefore the maximal total charge of a protein molecule in acidic or alkaline media is equal to the number of amino or hydroxyl groups, while at the isoelectric point, i.e., at complete ionisation of hydroxyl and amino groups, the charge is equal to the total number of both groups. Thus the charges of a protein molecule is more or less bound by counterions. A polyelectrolyte molecule in a semidilute solution can be regarded as a random walk of electrostatic blobs (Dobrynin et al. 1995). The blob charge of a polyelectrolyte not botmd by counterions usually is in the amount of several units. It can be presumed that at the isoe~ectric point the charges of different blobs possess opposite signs. As the total number of blol~s is rather high, the entire protein molecule
6 appears electro-neutral. The counterion bounding both in separate blobs and in the whole protein molecule is about 90 %, which corresponds to ionic micelles. Thus, the number of unbound charges of a protein molecule remains sufficiently large, and counts to tens or hundreds. The interaction between unbound charges has to result in strong repulsion between polyelectrolyte chains as shown by Klein & Luckham (1982, 1984). Based on the Gouy-Chapman theory Davies (1951, 1958) had derived an adsorption isotherm and an equation of state for charged surfactant molecules using an electric double layer model. The same model (DEL) was used by Borwankar & Wasan (1988), but they took the nonideality of the surface layer into account. Combining the results of Davies and Borwankar & Wasan with Eqs. (1.7), (1.10), (1.11), (1.13), and using the condition co0=c0z, one can transform Eq. (1.3) into
FI = -
RT[In(1- Fzcoz) + a(Fzcoz)2]+ ~4[RT,2 g R T c z } ,'/2rtchq~-l] p
it) E
-
-
(1.12)
where F is the Faraday constant, e is the dielectric permittivity of the medium, cz is the total concentration of ions within the solution, q~= zFt~/2RT, z is the number of unbound unit charges in the protein molecule, and W is the electric potential. Substituting the chemical potential by the electrochemical potential, the following expression can be obtained instead of the adsorption isotherm (1.4)
Kc(
0ifis
1-
0i
exp(2q~)
(1.13)
(f~)n,
_
The electric potential is determined by the surface charge density zFzF sh~ = (8eRTcz)1/2
(1.14)
Analysis of Eq.(1.12) has shown that for a usual 1:1 ionic surfactant at low bulk ion concentration, the approximate relation tp )~ 1 is valid (Fainerman 1991). This approximation leads to a linear dependence of H on F z in the electrostatic term of Eq. (1.12). For protein solutions, however, the situation is quite different. At high ion concentrations the Debye length
ae = (sRT/F2cx) in is small; e.g., for c x = 0.1 mol/1 the value of ae = 1.3 nm. This means that for protein solutions the DEL thickness can be smaller than the adsorption layer thickness. Therefore the concentration of ions in Eqs. (1.12) and (1.14) is just their concentration within the adsorption layer, which can exceed 1 mol/1 due to the ionisation of hydroxyl and amino groups, and the contribution of counterions. It follows from Eqs. (1.12) and (1.14) that for large c x the approximation tp _ 10 mN/m, only a small number of adsorbed molecules occupy an area exceeding (Oi "-O)min----2 nm 2. Therefore, the equilibrium adsorption layer is formed by almost
13 completely denatured proteins at low surface pressure, while for large surface pressure it is built by molecules in a natural state with a minimum surface area demand.
0,08 0,07 0,06 r
0,05 0,04 ._
0,03 0,02 0,01 0 0
10
20
30
40
50
60
70
2 C0 i , n m
Fig. 1.3. Dependence of the distribution function Fi/Fz on c0i for a protein solution (M = 24000, c01= 2 nm2, C0ma x -" 6 0
am2, (X- "
2
and ael = 600) at 1-I = 0.1 (1), 0.5 (2), 1 (3) and 5 mN/m (4).
The protein adsorption layer coverage remains very low for surface pressures FI around 20 to 30 mN/m if ael is sufficiently large (Fig. 1.2). The theoretical model of Eqs. (1.20)-(1.24) predicts a subsequent unrealistic sharp increase of surface pressure with weak increase of protein concentration, and a simultaneous slight increase of the adsorption. This contradicts experimental data which show that, starting from some protein bulk concentration, FI remains almost constant, while the adsorption continues to increase. This results in an increase in surface coverage which in turn leads to an almost complete saturation of the adsorption layer at high'protein concentration (Graham & Phillips, 1979b). Graham & Phillips had attributed these results to the formation of a second adsorption layer towards the solution bulk. Subsequently some attempts were made to apply this hypothesis to explain the fact that the surface pressure is independent of the adsorption in concentrated surface layers. A theoretical adsorption isotherm which agrees with the experimental data was derived by Guzman et al. (1986). Douillard & Lefebvre (1990) also employed the two-layer model of protein adsorption, which assumes that the composition of the first layer only affects
14 the surface pressure. It can be argued that a multilayer adsorption model is quite appropriate to describe protein adsorption at a solid surface. The self-consistent field theory developed by Leermakers et al. (1996) can be referred to as an example. For the water/air interface, however, at least for the globular HSA-type protein, statistical models do not indicate the possibility for the formation of a second layer, see e.g. Uraizee & Narsimhan (1991). The phenomenon discovered by Graham & Phillips was explained by Makievski et al. (1998) in the framework of monolayer adsorption of proteins, assuming that the inter-ion interaction parameter of the surface layer equation of state decreases with increasing adsorption, i.e., with increasing ionic concentration in the surface layer. Adsorption increase in the concentrated protein adsorption layer does not lead to an increase in the surface pressure. We believe that this effect is related to the formation of two-dimensional aggregates rather than a second layer, which, however, cannot be completely excluded. The results of Graham & Phillips (1997c), plotted as surface pressure FI versus area per adsorbed protein molecule (protein mass) A, show the characteristic behaviour of an insoluble monolayer which exhibits a transition region from a liquid-expanded to a liquid-condensed state, i.e., an inflection point and almost horizontal portion between the inflection point and the collapse point exist. It is therefore quite natural to explain the phenomenon by a 2D transition in the protein adsorption layer. Equations (1.20), (1.21) and (1.23) can be generalised for the case when a 2D aggregation of the proteins in the monolayer lakes place. To do so, we proceed first with a simplification of these equations, noting that for large surface pressures only state 1 exists as it follows from Eq. (1.24). Then, it follows from Eqs. (1.23) and (1.24) that F z = F 1 and coy = col. Theoretical models which assume aggregation in adsorbed and spread (insoluble) monolayers were proposed by Ruckenstein & Bhakta (1994), Israelachvili (1994), Ruckenstein & Li (1995), Fainerman et al. (1996b) and Fainerman & Miller (1996a). One can easily modify Eqs. (1.20) and (1.21) for monolayers comprised of monomers and aggregates by expressing the protein adsorption F z as the sum of the adsorption of monomers in state 1 (F 1) and of aggregates (mmers) Fz =
F m.
F 1+
Therefore, the adsorption of protein expressed in terms of kinetic entities is
F m.
On the other hand, the measured total adsorption recalculated in terms of
15 monomers is defined as Fs = F 1 + mF m . The equilibrium between aggregates and monomers in the surface layer can be described by the relation
F m = Fl ~ , ~ )
(1.28)
which has been derived by Fainerman & Miller (1996a) from the mass action law in the framework of a quasi-chemical aggregation model. Here F c is the critical adsorption of protein aggregation in the surface layer, that is, the value of adsorption at which the surface pressure attains the value 1-I~. For the isotherm plotted in I-I- A co-ordinates, this adsorption value corresponds to the inflection point. Therefore, for the total adsorption defined above, one can write: m-1
(1.29)
I-'; = r 1 + m F m = r 1 d- m F 1
(1.30)
For m ~ 1 (which seems to be the case) very simple relations follow for F 1, F m and cox, namely F 1 _---Fc, F m -- 0 and cox
= (o)11-";/1-"c ).
Note that mF m r 0. It is clear that
1-"m =
0 also for
I-" 1 < F c.
Therefore, for F x > F c the equation of state for the surface layer and the adsorption isotherm of the protein solution can be presented in the form"
l-I= -~,RT[F~ l n 0 - F: co, ) - a ~,co~F~ ]
(1.31)
bc =
(1.32)
Fzcol 1 - Fxco~
It is seen that the adsorption isotherm (1.32) predicts an increase in protein adsorption for F z > F c. For example, the values F c - 2.0-2.5 mg/m 2 found for HSA and 13-casein correspond to a monolayer coverage of Fcco1 =0.5-0.6. Therefore, the adsorption for the monolayer at
16 maximum coverage (provided that minimum area per protein molecule remains unchanged) counts towards 4-5 mg/m 2. Retaining only the leading term in the expansion of the logarithm in Eq. (1.31), one obtains H = const for Fr
> r c.
Retaining two terms in this expansion, one can
show that the difference between the surface pressure H at F z > Fr and that at Fr = F c (i.e., when H = Hc) can be expressed as
H=H
+
RTFco~ 1 2
~1 x - F + )
(1.33)
Noting that the area per protein molecule in the surface layer can be expressed as A = 1/F~, and using (1.31) one obtains
H=
__RT A
co~ _
- c~ A-~-ln 1 - ~ -
col ael
(1.34)
or approximately (again retaining two leading terms in the expansion of the logarithm)
H=Hr
2A~-
-1
(1.35)
where A c = 1/Ft.
1.2. Adsorption kinetics The equilibrium states of adsorbed protein molecules as described above may change under certain conditions.
In fact, an evolution of the equilibrium states occure if the adsorption
process is extremely slow. In addition, the reconstruction process of the molecular states within the surface will influence the adsorption kinetics of protein. The state of the protein molecule within the solution bulk depends on the structure of the molecule and properties of the solvent, such as pH value and ionic strength. It can be assumed generally that a certain set of molecular conformations in the bulk exist, which differ from one another in the coi values at the moment of initial contact with the surface. Therefore the total bulk concer,tration c of a protein is the sum of concentrations ci (c = Zci), which correspond to
17 various conformations of the molecules in the bulk. The equilibrium composition of the adsorption layer (Fi/Fz), the surface layer, is controlled by the surface pressure. In general, the composition of the surface layer does not coincide with that of the bulk phase; therefore the mi values in the surface layer will differ from the corresponding bulk values. This will lead to a reconformation of states within the adsorption layer. We can consider Fig. 1.3 as an example. Assume that the flow of protein molecules from the solution bulk is comprised mainly of the states possessing mi = 20 nm z. At I-I = 0.5 mN/m the most probable state for the equilibrium composition of the surface layer is also the one with mi = 20 nm 2. Therefore at 1-I - 0.5 mN/m the conformation of the adsorbed molecules within the surface layer will actually remain unchanged. However due to the subsequent increase of the adsorption and corresponding increase of surface pressure, both the relative and absolute number of the equilibrium states with ( O i - - 2 0 n m 2 will be continuously decreased. For example, at 1-I = 1 mN/m the most probable state will be the one possessing mi = 10 nm 2. Therefore both the molecules adsorbed earlier, and the new molecules with mi = 20 nm 2 which had just approached the surface, will undergo a reconformation within the surface layer: some portion of their segments will have to desorb. It is to be noted that for the initial state of the protein molecule within the surface layer a more realistic value of mi would be between.m1 and 2ml. This means that according to the model for small FI values all adsorbed molecules will undergo a denaturation within the surface layer. The reconformation of states of adsorbed molecules which initially possess, i.e., the i th state, can be represented schematically as: k; k;, ri_ 1 ~ r i ~ ri+ 1
(1.36)
where the superscript '+' or '-' at the kinetic rate constants k denote the forward or backward reaction, respectively. The mass balance equation for the ith state of the adsorbed molecules can be given in the form:
dr~ dt
- -Fi(k 7 + ki+,)+ Fi_,k + + Fi+,kT+, + I i
(1.37)
18 where Ii is the diffusion flux of the ith state molecules from the solution bulk. Therefore the variation rate of the adsorption for this ith state depends on its reconformation rate due to the decrease of O)i by Ao~, i.e. the rate for the closest conformations which differ from the considered one by Ao~, and the diffusion flux of this state from the solution bulk. For the description of the adsorption kinetics, the model of discrete molecular states within the surface layer seems to be more suitable. According to Fig. 1.3, the process of surface denaturation of proteins, that is, the increase of o~i with respect to the initial value, takes place for very low surface pressures. At low FI the process of protein adsorption seems to be controlled by diffusion (Miller 1991). The experimental data presented by Benjamins et al. (1978), Paulsson & Dejmek (1992), Ghosh & Bull (1963), Graham & Phillips (1979a), Kalischewski & SchOgerl (1979) and de Feijter et al. (1987) support the diffusion model for at least up to values of FI < 2 mN/m. From the results published by Ghosh & Bull (1963), Kalischewski & Schtigerl (1979) and de Feijter et al. (1987) it could be deduced that the time t* at which ~ begins to decrease, and the protein bulk concentration in the range from 0.001 to 0.05 g/l, are related to each other by the expression c2t*= const. The latter follows from the simplest diffusion kinetics equation valid for FI ~ 0 (Miller 1991):
FE(r~__,0) = 2
(1.38)
where D is the diffusion coefficient, and t is the time. It can thus be supposed that in low concentrated protein solutions the surface denaturation process has enough time to be completed, and therefore the composition of the adsorption layer at FI < 2 mN/m corresponds approximately to the equilibrium composition. Further reconformation processes of the states within the surface layer depends, according to Fig. 1.3, on the desorption of segments which were adsorbed previously. One can assume as a first approximation that only backward reactions in Eq. (1.36) affect the value of dFi/dt
dr~
dt-= Fi+lk~-+l-Fik ~-+ I i
(1.39)
19 Assuming that Fi = Fi~ AFi and Fi+1 = Fi+l~+ AFi+l, where the Fi values with superscript '0' refer to the equilibrium state (at equilibrium the relations dFi/dt = 0 and Ii = 0 hold), and for small deviations from equilibrium we obtain from Eq. (1.39) dAFi dt
_
A17"i+lk~-+l-Al-'ik~-+ I i
(1.40)
An important result of the theory of equilibrium adsorption of proteins is the fact that the kinetic constant of the backward reaction for any i th state can be expressed via the kinetic constant for any particular state, say, i = n. It follows from Eqs. (1.24) and (1.40) that ia ( (n-1)YIcol] k~- = - - k ~ e x n RT
(1.41)
The kinetic constants for the forward reactions can be expressed similarly. As the constants k~ and k~- are interrelated via the adsorption equilibrium constant bi, and all bi in turn are related to b~, it follows that to describe the adsorption kinetics in the framework of the proposed model, in addition to the equilibrium adsorption characteristics (col, COma• a and bl) only one extra kinetic constant, say, k~, and the coefficient of the bulk diffusion of protein D would be required. An important practical result follows immediately from Eq. (1.41). One can see from Fig. 1.3 that for FI > 5 mN/m the adsorption layer is comprised mainly of the states with col < 2oi. In this case the adsorption rate will be determined by the transition of F2 (with o2 = 2ol) into F1, that is, the molecules from the solution can occupy the place at the surface if some molecules being in state 2 would transform into state 1, therefore making room in the adsorption layer. Thus if the adsorption is controlled by the process I-'2 ~ Fl, then assuming FI-- Fz (which is true within a narrow FI range), from Eqs. (1.40) and (1.41) one obtains dYI p( YICOl/ d t = k~ ex - - ~ )
(1.42)
where k0 is a constant. This equation is the well-known MacRitchie relation (1977, 1989, 1991), derived from experimental data. For a number of proteins the COl value in Eq. (1.42)
20 varies in the range of 0.5 to 2.5 nm 2 (MacRitchie 1991), which agrees with the estimates of o)1 as the minimum surface area occupied by a protein molecule in the adsorption layer, or the increment of molar surface area Am for the chains possessing limited flexibility. It is clear that protein adsorption in high concentrated solvents differs significantly from protein adsorption in low concentrated solvents as described above. During protein adsorption in high concentrated solvents surface denaturation cannot be completed because the rate of any increase in 0~i is limited, and thus there is not enough room in the surface layer (W0stneck et al. 1996a). In contrast to low concentrated solvents where unfolding of the molecule within the surface is followed by a refolding process, almost no surface denaturation takes place in high concentrated solvents, and the composition of the dynamic surface layer is similar to the initial conformation distribution of the adsorbed molecules. This view explains why the shear elasticity and viscosity for 13-1actoglobulin adsorption layers formed at low protein concentrations were found to exceed those measured at larger concentrations, while the surface tension of the solutions remains constant (Kr~igel et al. 1995). One can expect that many unusual properties of the dynamic adsorption layers of proteins can be explained by protein molecule processing during the reconformation at the surface.
1.3. Experimental studies of model biological liquids The surface tension isotherms and dynamic surface tensions of HSA and bovine serum albumin (BSA) are essentially the same and were studied in a number of publications (Peters 1985, Graham & Philips 1979a, 1979b, 1979c, Lassen & Malmsten 1996, Serrien et al. 1992, Suttisprasit et al. 1992, Paulsson & Dejmek 1992, Dussaud et al. 1994, Turro et al. 1995, Hansen & Myrvold 1995, Boury et al. 1995, Miller et al. 1993 and Tripp et al. 1995). The dynamic surface tensions for other protein solutions (lysozyme, myoglobin, 13-1actoglobulin, 13-casein, ribonuclease, etc.) were investigated by Graham & Philips (1979b), Serrien et al. (1992), Paulsson & Dejmek (1992), Douillard et al. 1994), Xu & Damodaran (1994), Kr~igel et al. (1995), WOstneck et al. (1996b), Hermel & Miller (1995), MacRitche (1989) and Clark et al. (1995). Very low HSA or BSA concentrations (ca. 0.01 g/l) decrease the equilibrium surface tension at pH 7 to values of 50- 52 mN/m. Increasing the concentration of HSA or BSA from 0.01 to 10 g/1 decreases the equilibrium surface tension only by an additional 2 to 4 mN/m. It
21 has to be noted that for low-concentrated HSA solutions, the time required to attain the adsorption equilibrium is 20 to 30 hours, while for concentrated solutions this time is only a tens of a second or a few minutes . Most studies of dynamic surface tension with HSA solutions were done at concentrations of less than 1 g/l, while no systematic studies were carried out at concentrations reassembling blood (approximately 35 to 50 g/l).
73[] [] O DD
68-
9
~
9
63-
9 AA o
~ 9
[]
%
9 O0
AA
0(30
%
% c~ 9A OQ
58-
53 1
I
r
I
I
10
1 O0
1000
10000
I
100000
t, [s] Fig. 1.4. Dynamic surtace tension c tor HSA solutions at various concentrations: 2.10"8mol/l (m), 5.10.8 mol/1(+), 10 -7 mol/1(O), 5.10 -7 mol/1(A),10"6mol/l (D), 10.5 mol/l (o). To verify the equations of state and adsorption isotherms as derived above, experimental studies on both dynamic and equilibrium surface tensions for HSA solutions were performed using the ADSA method (Rotenberg et al. 1983, Cheng et al. 1990). The dynamic surface tensions for HSA solutions at various concentrations are shown in Fig. 1.4 (Makievski et al. 1998). It is seen that within the time range of up to 4 hours, equilibrium is achieved only with HSA concentrations > 10 -6 mol/1. This is in agreements with data presented by Gonzalez & MacRitchie (1970) who studied the BSA whose structure and properties are similar to those of HSA. To obtain estimates for the equilibrium surface tensions for less concentrated HSA solutions the curves cy = o(t) were extrapolated to t ~ oo. The dependencies of o as functions of t 1/2 are presented in Fig. 1.5.
22 For the mixed adsorption mechanism the derivative do/dt -1/2 is defined by the relationship obtained by Fainerman et al. (1994)
do dt -'/2 -
RTF~ c
n 4-D
)~ RW_____~
(1.43)
+ cJ3t1/~
where 13 is the adsorption rate constant. For 13 ~
oo which corresponds to the diffusion
adsorption mechanism, or for t ~ o o the second term in the right hand side of Eq. (1.43) vanishes, and therefore this expression transforms into the known relationship presented by Joos and Hansen (see Chapter 2). In fact, the curves presented in Fig. 1.5 possess a linear part in the large t region (which corresponds to small t ~/2 values), and therefore the point of intersection with the abscissa axis corresponds to the equilibrium surface tension. However, the experimental values of (do/dtl/2)t_,|
estimated from Fig. 1.5 are approximately 100 times
higher than those calculated from the Joos-Hansen formula.
" " 70
ee
9
AAA
;~ 60
**** DO
o
9
0
9
9
[]
[]
O
[]
55 |
50 ~0
,
,
~
0,01
0,02
0,03
t
~ 0,04
9 0,05
-I12 -I12 ,s
Fig. 1.5. Dynamic surface tensions g of HSA solutions as a function of tm for different bulk concentrations, c = 2.10.8 mol/l (11), 5.10"s mol/l (o), 9.10.8 mol/l (A), 2.10-7 mol/l (@), 10.6 mol/l (D). Thus in case of HSA the range within which a diffusion adsorption mechanism takes place, can be estimated from Eq. (2.19) as t ~/2 < 10 .4 S"1/2, that is, t > 108 s. In this t range a considerable decrease of the slope of o = o (t "1/2) is observed. However, this does not affect significantly the estimated position of the intersection point with the abscissa as this range is rather narrow. On
23 the other hand, if the second term in the right hand side of Eq. (1.43) contributes mainly to the derivative, the first term can be neglected, and therefore an extrapolation dcr/dt 1 is more justified. The data presented in Fig. 1.5 are replotted in Fig. 1.6 in ~ versus t l coordinates. One can see that also in this plot a linear part exists for t---~.
73
"jim
II
o
9
68
63
g-
[]
[]
58
53
--
0
1
0,001
[]
[]
1
0,002
T
0,003
Y
-
-
0,004
I
0,005
Fig. 1.6. Dynamic surface tensions of liSA solutions plotted as a function oft]: c = 2.10.8 mol/l (11), 5.10.8 mol/1 (A), 9.10.8 mol/1 (o),
2 . 1 0 -7 mol/l (O), 10 -6 mol/l (D).
The equilibrium surface tensions obtained from the two extrapolation procedures discussed above (t -1/2 and t -1 at t ~oo) were found to be close to one another, with differences generally smaller than 0.5 mN/m. In the following the average values between these two extrapolations were used. In Fig. 1.7 the experimental equilibrium surface pressure isotherm of HSA at pH 7 at the solution/air interface is plotted versus the initial HSA bulk concentration. It is to be noted that for HSA concentrations > 10 -7 mol/1 our data agree well with data for BSA presented by Gonzalez & MacRitchie (1970), Feijter et al. (1978), Ward & Regan (1980), ~'ornberg & Lundh (1981) and Graham & Phillips (1979b). In contrast, for HSA concentrations < 10 -7 mol/1 the values obtained from the ADSA experiments were
lower compared to the
results reported by Graham & Phillips (1979b). One likely explanation for this discrepancy is a potential decrease of the protein concentration within the drop due to the adsorption of protein molecules at the drop surface (Makievski et al. 1998).
24 The theoretical curves presented in Fig. 1.7 were calculated for the following set of parameters using Eqs. (1.20)-(1.24), (1.32) and (1.33)" o~l 03max - - 8 0
nm 2, Ao3 - -
~1,
ael--
-
(Omin -- 4 0
nm 2
(area per HSA molecule),
320, c~ - 0 and bl = 2.107 l/mol. These parameter values agree
remarkably well with the data presented by MacRitchie (1977, 1991), Murray & Nelson (1996) and Peters (1985). In particular, the minimum area per BSA (or HSA) molecule within the monolayer is in fact 40 to 50 nm 2. In spread BSA monolayers the increase in surface pressure becomes appreciable when the area per protein molecule decreases to about 150 nm 2, which corresponds to a monolayer coverage of approximately 20 %, see Fig. 1.2.
2520-
_t~
9
600
IO-L 5 -
1,00E- 10
1,00E-08
1,00E-06
1,00E-04
c [mol/l] Fig. 1.7. Experimental equilibrium surface pressure isotherm of HSA for pH 7 plotted as a function of the initial HSA concentration c, solid line - theoretical adsorption isotherm calculated from Eqs. (1.20)(1.24), (1.32) and (1.33). It has to be noted that for Aco - col a variation of
03max
in the range from 40 to 200 nm 2 does not
effect the theoretical dependencies of FI and Fz on c. For HSA at approximately 1 mg/m 2 and a total concentration of ions within the surface layer of 2 mol/1, with the total number of aminoacid groups in the HSA molecule equal to 585 (Peters 1985)the adsorption layer thickness is 4 rim.. Assuming the minimum free charge of an albumin molecule with z ~ 20 (Peters 1985), one can estimate the value of ael theoretically, see Eq. (1.17), which agrees with the value obtained from fitting the isotherm to the experimental data. The minimum area of an adsorbed HSA molecule corresponds to the three-domain molecular structure, with each domain being
25 comprised of 9 loops connected by sulfide bridges. At pH 7 the size of such molecule is 14 x 4 x 4 nm. This configuration is possibly independent of H and Fz, meaning that HSA molecules do not undergo denaturation at a liquid/gas interface. The FI - A isotherm for a BSA adsorption layer, as reproduced from Graham & Philips (1979c), is shown in Fig. 1.8. Theoretical
curves were calculated
from Eqs. (1.20)-(1.24)
for
A > A c = 0.5 m2/mg, and from Eq. (1.35) for A < A c. Again, the parameters of these equations were
O)max =
80 nm 2
(per protein molecule), (Omin =
At0 = 40
nm 2, (z = 0 and aei = 320. It is
obvious that the theoretical curves agree well with the experimental 1-I - A isotherm.
2520-
"''-..
lO
0,0
0,5
1,0
1,5
2,0
A [m2/mg]
Fig. 1.8. Dependence of surface pressure on the area per 1 mg of BSA in the surface layer. A - data from Graham & Phillips (1997c) for adsorbed layers, dotted line - data from Murray (1997) for spread layers, solid line - calculated for A > 0.5 mVmg from Eqs. (I.20)-(I.24), and for A < 0.5 mVmg from Eq. (1.35). Moreover, for the same parameter values and concentrations lower than the critical value, c < cc = 1.4.10 .3 g/1 (2.10 -8 mol/1), {cc is the bulk concentration at which the aggregation in the surface begins} the model corresponding to Eqs. (1.20)-(1.24) describes satisfactorily the experimental H - c and F z - c isotherms of BSA as reported by Graham & Philips (1979b). Figure 1.8 also shows the experimental isotherm for a spread monolayer of BSA (Murray, 1997). This dependence is seen to be rather similar in its details to that characteristic for the adsorption layer. There exists a kink point at H c = 18 mN/m, and an increasing portion for area
26 values lower than A c. Other conclusions can be drawn for the protein [3-casein. Graham & Phillips (1979b) have generated isotherms for surface pressure and adsorption independently using the Wilhelmy plate method in a Langmuir trough for measuring surface tensions and radioactivity and ellipsometry methods for measuring adsorption of aqueous solutions of i3-casein. They found that the two experimental isotherms correspond satisfactorily to the theoretical model when using the following parameters: et al. 1996). The value about
8min =
(-OI =mo,)=
f-Omax ---- 8 0 nlTl 2
6 n l I l 2,
O,)max =
80 nm 2, Ot = 0 and a~ = 100 (Fainerman
corresponds to a minimum adsorption layer thickness of
0.75 nm for a completely denatured [5-casein molecule. These results agree with
measurements of the mass of adsorbed protein from the concentration decrease inside a drop at the moment when the decrease of surface tension begins to occur (Miller et al. 1998a). The value o~l = 6
nm 2
corresponds to the maximum adsorption layer thickness of
8max --
6.7 nm that
agrees well with experimental data of Atkinson et al. (1995). It can be stressed that all three independent experimental data sets obtained by Graham & Phillips, i.e. Fx = Fx(c), FI = H(c) and 8 = 8(c), and also the corresponding derived dependencies, e.g., 1-I = H(Fx) or 8 = 8(Fx), agree satisfactorily with the multiple molecular state model for protein molecules at the surface for the same set of four main parameters of Eqs. (1.20)-(1.24).
1.4. Influence of additives
The addition of inorganic electrolytes, urea, simple carbohydrates, ionic or non-ionic lowmolecular surfactants, and variations in the pH of a solution, affect significantly both equilibrium and dynamic surface tensions. These additives, including the surface active molecules, influence not only the properties of the solution itself, but they mainly effect the properties and structure of HSA molecules, resulting in binding to or ionisation of amino acid groups, interaction within polypeptide chains, variation of the HSA molecular conformation in the bulk and in the surface layer. It has to be noted here that data concerning these effects are still scarce and often contradictory. It is known that the addition of inorganic ions (K +, Li +, Na +, 2+
2+
2+
3+
Ca , Mg , Fe , Fe , CI, F, HPO42-, PO43-, etc.), usually surface inactive substances, results in an increase of the surface tension. This increase of surface tension of biologic liquids due to
27 the increase in concentration of inorganic salts can be significant in the short time range, when the adsorption of proteins and other surfactants is relatively small or even negligible. In contrast, for medium and long surface lifetimes adsorption results in a decrease of surface tension. For example, the addition of 0.1 mol/1 NaC1 to a BSA solution decreases the surface tension in the medium and long time range (Kalischewski & Scht~gerl 1979). The effect of the solvent properties on dynamic surface tensions of BSA solution was illustrated by Paulsson & Dejmek (1992). When distilled water was replaced by synthetic milk ultrafiltrate (SMUF, pH 6.6, ionic strength 0.08), the surface tension of a BSA solution at a concentration of 0.1 g/1 after a surface lifetime of approximately 50 s had decreased to 72 mN/m for water and to 60 mN/m for the SMUF solution. It was shown by Fainerman & Miller (1996b) and Joos & Serrien (1989) that fructose (and, similarly, glucose and saccharose) promotes the structuring of water molecules, while urea destroys this structure. These effects influence significantly the adsorption activity of lowmolecular weight surfactants. For example, the addition of fructose leads to a decrease of the dynamic surface tension for both short and long surface lifetimes, while the addition of urea results in an increase of cr(t). On the other hand, the effect of these additives on dynamic surface tension of protein solutions is not restricted to the variation in the structure of the solvent. The addition of urea leads to a denaturation of BSA or HSA both in the bulk and at the surface, which results in a significant decrease of the surface tensions (Serrien et al. 1992). The pH of the solution influences the secondary structure of protein molecules (Peters 1985), and can directly affect its adsorption activity (Hermel & Miller 1995). The decrease of pH with respect to its initial value of 7.5 leads to an appreciable decrease in surface tension of HSA (Hansen & Myrvold 1995) and other proteins (W~stneck et al. 1996b). The relative increase of the pH values also results in an increase of adsorption activity for concentrated HSA solutions, but this effect is less pronounced than that corresponding to a similar decrease of the pH value. The effect of low molecular weight non-ionic surfactants (alcohols, acids, oxyethylated ethers, etc.), on dynamic surface tensions of HSA and other proteins depends on their concentration and adsorption activity. For example, the effect of ethanol on the conformation of HSA or BSA
28 in solution leads to a significant decrease in its adsorption activity (Dussaud et al. 1994); this effect, however, can be overcompensated by the adsorption of ethanol, and the surface tension can therefore decrease. Oxyethylated surfactants, for example TWEEN 20 which possess higher surface activity (Kr/igel et al. 1995), produce almost no effect on the surface tension of [3-1actoglobulin solution at short lifetimes, but decrease significantly the equilibrium surface tensions. However, no definite predictions can be made concerning the effect of ionic surfactants (like sodium alkyl sulphates) on dynamic surface tensions, because in this case the inter-ion interaction between surfactants and proteins can result in the formation of protein/surfactant complexes (Turro et al. 1995). For very small additions of ionic surfactants (100 times lower than the protein concentration) an increase in the surface tension of the mixture occurs, and for relatively high additions of the same surfactant the surface tension generally decreases, while in some concentration regions anomalous surface tension behaviour of the mixture was observed (Wiistneck et al. 1996b). For mixtures of surfactants such effects were predicted and observed by Fainerman & Miller (1997). The experimental and theoretical study of the adsorption behaviour of mixtures of the globular protein (HSA) and a non-ionic surfactant (decyl dimethyl phosphine oxide, CI0DMPO ) was carried out by Miller et al. (1998b). This particular system was chosen because it is a good model system for a theoretical analysis:
it can be described in the framework of known
theoretical models, and studies of the adsorption properties of such mixtures promote insight into the mechanisms goveming the variations of surface active characteristics of serum caused by various diseases. The adsorption of the proteins and their mixtures with surfactants was characterised by dynamic surface and interfacial tension measurements using the axisymmetric drop shape analysis (ADSA). The standard deviation of the ADSA method in these studies was 0.2-0.3 mN/m. The surface tension measurements of C10DMPO were performed using the tensiometers MPT1 (maximum bubble pressure method) and TEl (ring method), all manufactured by Lauda, Germany. The MPT1 device and measuring procedures are described in Chapter 2.
29
75 70 65
~ 6o ;~ 55 1 b
50 45 !
~
40-
T 0
~
5000
~
10000
15000
20000
t Is] Fig. 1.9. Dynamic surface tensions of HSA/C 10DMPO mixtures at various surfactant concentrations: 110 "9 (O), 110 -8 (A), 410 "s (A), 710 -8 (~3), 110 -7 ('r
210 -7 (*), 410 "7 (O), 710 "7 (11), 1.10 -6 (x)
mol/cm 3
75
ImVN
*
ii
65 xxX
55
Z
45
35
I' '
0
000
-
-
V'~.,IV
1
"
~
v
2
,..A-,
3
x X x
Oo
xX
9 00
0
v
4
5
t -I/2 [s -1/2] Fig.1.10. Dynamic surface tension of CIoDMPO solutions plotted as functions of t "~'z , vertically dotted line t = 100 s; concentrations: 110 "7 (O), 210 -7 (In), 5.10 -7 (A), 110 -6 (x), 210 "6 (0), 510 "6 (O) mol/cm 3
30 The dynamic surface tensions for HSA mixtures (concentration 10-7 mol/1) with C10DMPO for various surfactant concentrations are shown in Fig. 1.9. These have to be compared with the dynamic curves for pure HSA solutions (Figs. 1.5, 1.6), and pure C10DMPO solutions (Fig. 1.10), respectively. The dynamic curves for the surfactant are plotted in the coordinates c~ versus t l/z. The theory predicts that in these coordinates a linear dependence should exist at t > 100. This is clearly supported by experimental data. The intersection of the linear portion of the curves with the ordinate corresponds to the equilibrium surface tension. Note that for C~0DMPO solutions the time necessary for the equilibrium to be attained is rather short. The time value t = 100 s is marked in Fig. 1.10 by the dotted line. It is seen that the dynamic surface tensions for all the concentrations studied at this time moment differ from the equilibrium values by less than 1 mN/m. For HSA solutions the dynamics of surface tension decrease is rather different. Thus, in all the mixtures studied, preferential adsorption of CI0DMPO takes place first, followed by the adsorption of HSA. Therefore, the dynamic curves shown in Fig. 1.9 can be considered to consist of two sections: the first (t < 200 s) corresponding to the adsorption of C10DMPO, and the second (t > 200 s) - to the HSA adsorption. It is seen from Fig. 1.9, that for the concentrated (2-10 -4 mol/1 and higher) CIoDMDO solutions, the adsorption of HSA is almost absent, while for CIoDMPO concentrations c < 10.5 mol/1, only HSA is adsorbed from the solution. The equilibrium surface tension isotherms for CIoDMPO without and mixed with HSA are shown in Fig. 1.11. It is seen that for c > 10-4 mol/1, the two isotherms are almost indistinguishable. This also shows that the adsorption of HSA for the higher CIoDMPO concentrations is negligibly small. We conclude that the composition changes in the surface layer is rather sharp within a narrow range of C10DMDO concentration. This view is supported by the analysis of the curves of shear viscosity of mixed monolayers as shown in Fig. 1.12.
31
80
--
70-
i
E ;~60-
50-
40
........... -7
I. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . -6
-5
-4
I
-3
-2
1og c [mo 1/1] Fig. 1.11. Equilibrium surface tension isotherms for individual C10DMPO solutions ([2, solid line) and mixed C10DMPO/HSA solutions for the concentration 10 -7 mol/l (A), dotted line - equilibrium surface tension of pure HSA solution of c = 10-7 mol/l averaged over 6 measurements (~ = 57+ 1) mN/m.
100
-
80-
60-
Z K--
40
20
0 0
50
100
150
200
250
t [min]
Fig.l.12. Shear viscosity for surface layers of mixed solutions of HSA (c = 10 -7 mol/l) and C10DMPO at various surfactant concentrations" 10 -6 (m), 5.10 -6 ([3), 10 -5 ( 0 ) , 210 .5 (4"), 410 .5 (A), 710 .5 mol/l (A); the solid line corresponds to the surface shear viscosity of pure HSA, according to Miller et al. (1998b)
32 It is seen that for C10DMPO concentrations c _< 2.10 5 mol/1, the monolayer possesses a rather high viscosity, characteristic for pure HSA solutions. However, already at a concentration of 7.10 .5 mol/1, the shear viscosity decreases sharply almost to zero, which is characteristic of surfactant solutions. Therefore, both tensiometric and rheologic studies indicate that the compatibility of HSA and CIoDMPO in the mixed monolayer is very poor, in contrast, for example, to the mixtures of surface active homologues. For such mixtures no range of 'components antagonism' exists, that is, the addition of a second component always results in an extra decrease of surface tension for the mixture. This fact can be theoretically explained easily. It follows from the generalised Szyszkowski-Langmuir equation for the mixture of two components, 1 and 2, that RT In 1 + b2c2 ) AFI~2 = co 1 + b~cl
(1.44)
where AYI~2 is the extra decrease of surface tension for the solution of component 1, caused by the addition of component 2, e0 is the partial molar area of the surfactant, b the adsorption equilibrium constant. It is seen from Eq. (1.44) that, except for blC 1 >~b2c2, an addition of the second component leads to a substantial decrease of the surface tension of the mixed solution. Recalling Fig. 1.11, one sees that for CIoDMPO concentrations in the range c - (10 -5 - 10-4) mol/l, the surface tension of mixtures exceeds that of the individual HSA solution. (For CI0DMPO concentrations lower than 5.10 -7 mol/1, the surface tension of the mixtures was equal to that of the pure HSA solution). For concentrations in the range from c = 4.10 .5 mol/1 to 10 4 mol/l this surface tension excess amounts only to 1-1.5 mN/m. However, for c < 4-10 .5 mol/1, the surface tension of mixed solution exceeds that of the pure HSA solution by 3 to 4 mN/m. The theoretical model of Eq. (1.44) is unable to explain the increase of surface tension: an anomalous increase of the surface tension in the mixture of homologues, (that is, negative values of AFI~2) cannot follow from Eq. (1.44) by the addition of the second component. The hydrophobic interaction of C10DMPO hydrocarbon tails with HSA polypeptide chains can, in principle, lead to a hydrophilisation of the protein molecule. This effect, however, cannot be significant in our case, when only 10 molecules of C10DMPO (for c = 10-6 mol/1) exist in the
33 mixed solution per HSA molecule, which possesses 585 amino acid groups. This anomalous adsorption behaviour of a protein/surfactant mixture can be explained in the framework of equations of state (1.20) and protein adsorption isotherm (1.21) for a solution/fluid interface. Although 2 to 3 adsorption states may exist for HSA molecules in general, for surface pressures > 3 mN/m (c > 2.10 -8 mol/1) only one state persists. This state possesses minimal area per molecule O)min - - 4 0 nm 2. Therefore, in our case (c
= 1 0 "7
mol/1),
only one state has to be taken into consideration, resulting in an essential simplification of the model. Note that the adsorption of C10DMPO in absence of protein can be described quite well by the Szyszkowski-Langmuir equations. Thus, in a mixture of HSA (component 1) and C10DMPO (component 2), HSA exists in a single adsorption state.
VI = - RT [ln(1 - Fzmz)- ae,F2m~ ]
(1.45)
0) E
blC 1 =
FI (-1)1
(1 _ r~mz)~,/~ ~
(1.46)
F2m2
(1.47)
where Fz = F 1 + F 2. The average molar area of adsorbed components 1 and 2 can be expressed according to Eq. (1.6) by
mz =
Fl(O l + F 2 o ) 2
F~ + F 2
(1.48)
or
2
Flm~ + r2m2
(1.49)
(2) Z ~- rlfX)l -~-F20) 2
where the averaging in Eq. (1.48) and Eq. (1.49) was performed over the adsorptions and monolayer coverages, respectively. Equation (1.48) can be successfully used to describe mixtures of surfactants when the difference between the molar areas of the components does
34 not exceed one order of magnitude. Eq. (1.49) seems to be more appropriate for mixtures of a protein and a surfactant, where the % values differ by two orders of magnitude,. Note however, that for limiting cases, when a preferential adsorption of either of the two components takes place, the difference between the models of Eqs. (1.48) and (1.49) becomes negligible. For pure HSA solutions, the following values were found: col = 40 nm 2 (per molecule), b I = 2.1071/mol, and ael = 320. For pure C10DMPO solutions the corresponding values are co2 = 0.45 nm 2 (per molecule), and b 2 = 2.28.104 1/mol. The surface tension isotherm for CIoDMPO, calculated with these parameters from the Szyszkowski equation is shown in Fig. 1.11. The relation between the adsorptions of protein and surfactant can be derived from the adsorption isotherms of Eqs. (1.46) and (1.47):
:
F2co2
b,c,
(1 -
(1.5o)
b2c2
For a given ratio of the component concentrations in the solution bulk one can deduce from Eq. (1.50) that the portion of protein in the surface layer decreases sharply with the increase of the total adsorption
as coI ~ ~
The values calculated from Eqs. (1.45)-(1.49) for the
adsorption characteristics of HSA and C10DMPO as listed above, agree well with the experimental dependence shown in Fig. 1.11. This figure displays the anomalous increase of surface tension at low CIoDMPO concentration. Negligible adsorption of protein at high C~0DMPO concentration are predicted. For the limiting cases, one can derive simple analytical expressions for AFI. For low concentrations (adsorptions) of CIoDMPO, as col/coy~_=_1 and col/coz 10.4 cm) the excess pressure is reduced significantly due to high initial velocity of the gas. For capillaries for which the condition rc2/1 > 10.4 cm is satisfied, the value of Po does not exceed 0.5 % of P. Thus the hydrodynamic MBPM theory enables one to determine the conditions at which the aerodynamic component of the excess pressure Pd is minimised.
49 First theoretical calculations of the deadtime t d were performed with a Poiseuille approximation for the gas flow through the capillary by Fainerman (1979) 3
321q
o.
where r b is the separating bubble radius. The first term on the right hand side of Eq. (2.8) describes the gas expansion into an infinite space, while the second term corresponds to the capillary pressure in the growing bubble. The surface tension for a growing bubble cy* during the deadtime, which enters into the second term, is in fact unknown for surfactant solutions. The analysis performed by Fainerman (1990) had shown that for solutions the value of cy* in Eq. (2.8) is between the equilibrium value ooo and the dynamic value of cy for t = t l. Another important conclusion of this analysis is that a variation of cy* in the range o > ~* > cyoodoes not affect the t d value. This fact enables one to exclude o* from Eq. (2.8) by substituting cy instead. Thus in the Poiseuille approximation one obtains (Fainerman et al. 1994a) td=tb'~
1+~
where kp is the Poiseuille equation constant for the capillary not immersed into the liquid (L = kpP), L is the gas flow rate, P = Ps" P., and t b is the time interval between successive bubbles. A more rigorous deadtime theory was developed by Dukhin et al. (1996) and Koval'chuk et al. (1998b). These authors had shown that the corrections related to the nonstationarity of the gas flow through the capillary and to the effect introduced by the initial section of the capillary, do not exceed a few percent of the t d value calculated from Eq. (2.9). The hydrodynamic relaxation time lifetime:
th --
th
represents the sum of the first two components of the
tll + t12, that is, the sum of the times of forward and reverse meniscus motion. For
short capillary whose internal surface is hydrophobic, the liquid penetration depth h into the capillary is small, while for hydrophilic internal capillary surfaces h is of the order of the capillary radius. Therefore for hydrophilic capillaries the time interval th can contribute
50
significantly to tl. The values of tll and h for hydrophilic capillaries were first estimated by Dukhin et al. (1998). It was shown that for the aperiodic regime (K < 1) the value for h became 2- 3 % of the capillary length. Furthermore, the value for the forward meniscus motion time, tll, became 103s for long and narrow capillaries , and 10-Ss or less for short and wide capillaries ). It is to be noted that the value of h does not depend on the excess dynamic pressure in the system, P d = Ps -
PH - P ,
while the forward and reverse meniscus motion times depend strongly
on the ratio P/Pd" The larger the excess pressure, the lower is the meniscus hydrodynamic relaxation time. The value
PO
in turn depends on the capillary geometric characteristics and the
gas flow regime. For the aperiodic regime the value of t h is close to the lifetime in long narrow capillaries, while for short capillaries the inequality
t h 10 or capillaries possessing a hydrophobic internal surface (in both cases the liquid does not penetrate into the capillary after bubble separation) the pressure within the bubble throughout the whole lifetime stage remains constant Pb = (2eye/re)COS% = const
(2.18)
where eye and q), are the current instantaneous values of surface tension and contact angle during the lifetime stage (0 < t < tl). Equation (2.18) is the basic expression for the calculation for the relative dilation rate of the bubble surface. The following expression was derived by Makievski et al. (1994)
where cy is the dynamic surface tension for x = tl. It is seen that the dependence of 0 on ~ and z is rather complicated and does not obey the simple relation (2.16). Assuming that the bubble
54 surface area increase during the lifetime stage is relatively small (in fact, the largest possible area variation is from nr 2 for x - 0 to 2nr 2 for x = t 0, the finite variations of bubble surface area were analysed to estimate 0. It was shown that to within a reasonable accuracy the relative dilation rate and effective time can be expressed by Eqs. (2.16) and (2.17). The following expression for the constant ot was derived by Makievski et al. (1994) 2sin% ot = ~ 2 + sin%
(2.20)
where % = arccos(o/%), o 0 the surface tension of the solvent. For o = o 0 the value of ot is equal to 0, that is, the bubble surface is virtually non-deformed. For surfactant solutions with cy/cy0 < 0.8 we have ot ~ 2/3; therefore, similar to the case of a growing drop, tef= (3/7)t. It is to be noted that Eq. (2.20) is valid for short and wide capillaries, when t h 5.10 .5 cm) does not lead to errors in determining the ~ value. The employment of hydrophilic long capillaries however leads to significant errors in the measured dynamic surface tensions. The relation between the volume of separating bubbles and the measuring system volume affects the accuracy of dynamic surface pressure measurements as well. A ratio of Vs/V b > 5000 is recommended. In addition, the volume of the separating bubble must not be too large, otherwise the increased
td
values would restrict the MBPM applicability to weakly
concentrated solutions and long times. The studies of biological liquids have revealed that capillary parameters and properties produce significant effects in the precision and reproducibility of the data obtained. Best results were obtained with glass capillaries of narrow sections between 7 to 10 m m , and possessing internal diameter of 0.25 to 0.2 mm. We found that these capillaries are most useful for studying
59 biological liquids, because the liquid cannot penetrate into the capillary irrespectively Of the properties of the internal surface.
2.5. Analysis of t e n s i o g r a m s
The results of tensiometric studies of biological liquid are surface tensions at different surface lifetimes (Fig. 2.4). Such tensiogram for serum usually shows a relatively weak decrease of cr at short lifetimes, followed by a rapid decrease at t > 0.1 s. The shape of the tensiograms for other biological liquids show a wide variety: a sharp decrease of ty at t < 0.01 s, the existence of linear sections or one or two extrema, almost no dynamic features throughout the whole measured time range, etc. It is therefore a rather complicated problem to make comparisons between curves. To determine which dynamic tensiogram parameters are most informative, the asymptotic equations of the diffusion controlled adsorption kinetics theory at liquid interfaces have been employed.
74 72
~176176176 ~176 o Oo ~
00%
70 ~
68
66 % 64 0,01
I
t
t
I
0,1
1
10
100
t I [s]
Fig. 2.4. Dynamic surface tension cr of a blood serum sample as a function of surface lifetime h.
The dependence of surface tension on surface lifetime is governed by adsorption/desorption processes of surface active components at the liquid interface. At the initial time moment
60 (t = 0) the surface layer contains no excess of these components, that is, the adsorption is zero, and the surface tension of the solution is equal to that of the solvent, cy0. For most biological liquids, cy0 is close to the surface tension of water D 70 to 74 mN/m. In general, the adsorption rate and the rate of surface tension decrease are determined by the diffusion of surface active molecules towards the surface, and by restructuring processes of the adsorbed molecules within the surface layer (see Chapter 1). The basic equation of the diffusion controlled adsorption kinetics theory was proposed by Ward & Tordai (1946, Eq. 2.12). However, its application is rather cumbersome, because the solution of the resulting integral equation (a Volterra type nonlinear equation) requires additional thermod3,namic and kinetic relations (see Chapter 1). Thus for multicomponent biological liquids one can hardly expect at present any success in the rigorous solution of diffusion kinetics problems. Instead we believe that using asymptotic equations of this adsorption kinetics theory (Van den Bogaert & Joos 1982, Fainerman et al. 1994b, Miller et al. 1994, Hansen 1964, Rillaert & Joos 1982 and Bleys & Joos 1985), provides a more simple, and at the same time a rather informative method of the analysis of dynamic tensiograms. For the case of extremely short times (t --~ 0) a simple relation follows from the general Ward & Tordai Eq. 2.12. For multicomponent solutions this relation can be written as (Fainerman et al. 1994b)
do
= -2RT t--~0
c~ i=l
71;
where the subscript 'i' refers to any i th of the n components. The derivative on the left hand side of this equation (~-0 = [do/dt 1/2 ],-~0)is the slope of o as a function of t 1/2. As the values of diffusion coefficients for different components are of the same order of magnitude, it follows from Eq. (2.24) that this slope is roughly proportional to the total concentration of surface active components of the mixture. The data presented in Fig. 2.4 are re-plotted in Fig. 2.5 in the cy versus tl/2 coordinates.
61 74 ~-
72t~ ~'70-
t~ 6 8 - -
0 0
0
O0
66-
0 O0
0 O O
64 0
1
I
r
2
3
teff 1
/2
/2 [S 1 ]
-
t
4
5
Fig. 2.5. Dynamic surface tension of blood serum sample as a function of teff 1/2. Characteristics of the linear part are: Cyo=72,7mN/m, ~,o= 2,8 mN m-1 s-1/2. It is seen that a linear part of the curves for t--~0 exists in this case. The intersection point of this line with the ordinate axis corresponds to the surface tension of water (in our example ~0=72,7 mN/m). In general, the value of % is determined mainly by the salt composition of the biological liquid. Thus, comparing the dynamic tensiogram slopes in the co-ordinates cy versus t 1/2 one can draw conclusions concerning the total concentration of surface active components in the studied sample. One more important relation, following from the Ward & Tordai theory, is the so-called JoosHansen equation, which is valid for the case of extremely large surface lifetimes. This equation, generalised to multicomponent system, can be represented as (Fainerman et al. 1994b):
•
dcy I RT Fi2 / n dt-1/2 t-.oo = 2 -~-]c~i i i = ,
(2.25)
where F i is the adsorption for the ith surface active component. Here the derivative on the left hand side ( )~ = [dcy/ dt -~/2 ]t_.oo) is taken with respect to (1/2, and is calculated in the limit t --~ oo (that is, t 1/2 --~ 0). As for most surface active components the ratio Fi/ci is constant (the so called Henry constant K i = Fi/ci), and the sum on the right hand side of Eq. (2.25) is an approximate expression for the total adsorption of all mixture components with reference to
62 their adsorption activity K i. Therefore, comparing the values of the derivative ~L=(do/dt-1/2)t _~oo for various samples of biologic liquids, one can deduce information on changes in the adsorptions. The experimental dependencies presented in the Figs. 2.4 and 2.5 are replotted in Fig. 2.6 in the coordinates of Eq. (2.25). It is seen that the dependence 0 versus t 1/2 in fact possesses a linear part at t -1/2 --~ 0 (t ~ oo). The intersection point of this linear part with the ordinate axis corresponds to the equilibrium surface tension Goo (i.e., reduced to infinite time t ~ oo). This characteristic is extremely important; it is seen that it can be rigorously obtained only from the extrapolation of a dynamic tensiogram in the co-ordinates G versus t -1/2.
74 7-
72 ~
OO
O
O
O
O
O
O
O
7O +
~
68! 66
,,
64 / 62 i 1
t
I
I
t
2
3
4
5
tef? 1/2 [S "1/2 ]
Fig. 2.6. Dynamic surface tension of a blood serum sample as a function o f teff"I/2, the characteristics of the linear part are: g~o=62,9mN/m, k =11,2 mN m-~ s ~/2. In addition
to
the
tensiographic
parameters
mentioned
above,
namely
00,
0o0--03,
Lo = -(dG/dtl/z)t - o, ~" = (dG/dtI/Z)t- oo, we have used also the dynamic surface tensions at two other points of the tensiogram: 0~ for t = 0.01 s, and 02 for t = 1 s. The values 0 and Lo are characteristic for solvent properties and adsorption processes in the short lifetime range, while the value of 02 is indicative of the properties and processes in the medium surface lifetime range. These processes are governed mostly by the presence of low- and medium-molecular
63 weight surfactants in the composition of biological liquids, while the values of cy3 and ~ are controlled by the properties of high-molecular weight fractions of albumins and other compounds. As the result of this chapter one can conclude that the maximum bubble pressure technique is uniquely suited for studies of biological liquids. The methodology is well elaborated, experimentally and theoretically, and provides reliable dynamic surface tension data in a time interval important for these liquids. It turns out that particularly the time range of milliseconds up to seconds is extremely sensitive to the composition of blood, urine and other medically relevant liquids. The characteristic values which can be extracted from the complete tensiogram are sensitive to changes in the liquid composition, and hence carry information useful for diagnostic and therapeutic matter. The subsequent chapters will demonstrate this fact systematically.
2.6.
Summary
The maximum bubble pressure tensiometry is a modern and reliable tool to accurately measure dynamic surface tensions. It is shown how the measured physical values, capillary pressure as a function of gas flow rate, are interpreted as dynamic surface tension in function of the effective surface lifetime. The method gives access to data even in a time interval down to less than one millisecond. The method is theoretically well founded and all phenomena observed under the wide variety of experimental conditions can be described adequately by hydrodynamic theories. Also for viscous liquids, such as serum, the measured data are quantitatively understood. The analysis of the dynamic surface tension curves provides a number of characteristic values which are of great importance for medical research. Particular plots are discussed which give easy access to these characteristic values. The subsequent chapters 3 to 8 will describe which of the defined characteristic values correlate with biochemical data and thus are relevant as diagnostic tool and for monitoring the progress of medical treatments.
64 2.7. References
Austin, M., Bright, B.B. and Simpson, E.A., J. Colloid Interface Sci., 23(1967)108 Belov, P.T., Zh. Fiz. Khim., 55(1981 )302 Bendure, R.L., J. Colloid Interface Sci., 35(1971)238 Bleys, G. and Joos, P., J. Phys. Chem., 89(1985)1027 Borwankar, R.P. and Wasan, D.T., Chem. Eng. Sci., 41 (1986) 199 Bottomley, G.A., Austr. J. Chem., 27(1974)2297 Brown, R.C., Philos. Mag., 13 (1932) 578 Campanini, R., Swanson, A. and Nicol, S.K., J. Chem. Soc. Faraday Trans. 1, 72(1976)2638 Chang, C.-H. and Franses, E.I., Colloids Surfaces A, 100(1995)1 Cuny, K.H. and Wolf, K.L., Ann. Phys. Leipzig, 17(1956)57 Dukhin, S.S., Fainerman, V.B. and Miller, R., Colloids Surfaces A, 114(1996)61 Dukhin, S.S., Mishchuk, N.A., Fainerman, V.B. and Miller, R., Colloids Surfaces A, 138(1998)51 Dushkin, C.D., Ivanov, I.B. and Kralchevsky, P.A., Colloid Surfaces,60(1991)235 Fainerman, V.B. and Lylyk, S.V., Kolloidn. Zh., 44(1982)598 Fainerman, V.B. and Miller, R., J. Colloid Interface Sci., 175(1995)118 Fainerman, V.B. and Miller, R., J. Colloid Interface Sci., 178(1996)168. Fainerman, V.B., Colloids Surfaces, 62(1992)333 Fainerman, V.B., Kolloidn. Zh., 41 (1979) 111 Fainerman, V.B., Kolloidn. Zh., 52(1990) 921 Fainerman, V.B., Lylyk, S.V. and Jamilova, V.D., Kolloidn. Zh., 49(1987)509 Fainerman, V.B., Miller, R. and Joos, P., Colloid Polymer Sci., 272(1994a)731 Fainerman, V.B., Makievski, A.V. and Miller, R., Colloids Surfaces A, 87(1994b)61.
65 Fainerman, V.B., Makievski, A.V. and Joos, P., Colloids Surfaces A., 90(1994c)213 Fainerman, V.B., Makievski, A.V. and Miller, R.,. Colloids Surfaces A, 75(1993)229 Fainerman, V.B., Zholob, S.A., Miller, R., Loglio, G. and Cini, R., Tenside-Detergents, 33(1996)452 Feldman, I.N., Malkova, I.V., Sokolovskij, V.I. and Zaturenskij, R.A., Zh. Prikl. Khim., 53(1980)1594 Garrett, P.R. and Ward, D.R., J. Colloid Interface Sci., 132(1989)475 Geeraerts, G. and Joos, P., Colloids Surfaces A., 90(1994) 149 Hallowell, C.P. and Hirt, D.E., J. Colloid Interfaces Sci., 168(1994)281 Hansen, R.S., J. Phys. Chem., 60(1964)637. Hirt, D.E., Prud'homme, R.K., Miller, B. and Rebenfeld, L., Colloids Surfaces, 44(1990)101 Horozov, T.S., Dushkin, C.D., Danov, K.D., Arnaudov, L.N., Velev, O.D., Mehreteab, A. and Broze, G., Colloids Surfaces A., 113(1996) 117 Hua, X.Y. and Rosen, M.J., J. Colloid Interface Sci., 141 (1991) 180 Hua, X.Y. and Rosen, M.J., J. Colloid Interface Sci.,124(1988) 652 Huh, C. and Scriven, E.L., J. Colloid Interface Sci., 30(1969)325 Iliev, Tz. H. and Dushkin, C.D., Colloid Polymer Sci., 270(1992)370 Jaeger, F.M., Z. Anorg.Chem., 101 (1917) 1 Johnson, C.H.J. and Lane, J.E., J. Colloid Interface Sci., 47(1974)117 Joos, P. and Rillaerts, E., J. Colloid Interface Sci., 79(1981)96 Joos, P. and Van Uffelen, M., J. Colloid Interface Sci., 171(1995)297 Joos, P., Fang, J.P. and Serrien, G., J. Colloid Interface Sci., 151 (1992) 144 Kao, R.L., Edwards, D.A., Wasan, D.T. and Chen, E., J. Colloid Interface Sci., 148(1992)247 Keen, G.S. and Blake, J.R., J. Colloid Interface Sci., 180(1996)625 Kisil', I.S., Mal'ko, A.G. and Dranchuk, M.M., Zh. Fiz. Khim.,55(1981)177
66 Kloubek, J., Colloid Polymer Sci., 253(1975)754
Kloubek, J., J. Colloid Interface Sci., 41 (1972a)7 Kloubek, J., J. Colloid Interface Sci., 41 (1972b) 17 Kloubek, J., Tenside, 5(1968) 317 Koval'chuk, V.I., Dukhin, S.S., Fainerman, V.B. and Miller, R., J. Colloid Interface Sci., 197(1998)383 Koval'chuk, V.I., Dukhin, S.S., Makievski, A.V., Fainerman, V.B. and Miller, R., J. Colloid Interface Sci., 198(1998) 191 Kragh, A.M., Trans. Faraday Soc., 60(1964)225 Kwok, D.Y., Hui, W., Lin, R. and Neumann, A.W., Langmuir, 11(1995)2669. Lane, J.E., J. Colloid Interface Sci., 42(1973)145 Li, B., Geeraerts, G. and Joos, P., Colloids Surfaces A, 88(1994)251 Lunkenheimer, K., Miller, R. and Becht, J., Colloid Polymer Sci., 260(1982)1145 Lylyk, S.V., Makievski, A.V., Koval'chuk, V.I., Schano, K.-H., Fainerman, V.B. and Miller, R., Colloids Surfaces A, 135(1998)27 Makievski, A.V., Fainerman, V.B. and Joos, P., J. Colloid Interface Sci., 166(1994)6 Miller, R., Fainerman, V.B., Schano, K.-H., Heyer, W., Hofmann, A. and Hartmann, R., Labor Praxis, N9(1994)65 Miller, R., Fainerman, V.B., Schano, K.-H., Hofmann, A. and Heyer, W., Tenside-Detergents, 34(1997)357 Miller, R., Hofmann, A., Schano, K.-H., Halbig, A. and Hartmann, R., Tenside-Detergents, 28(1992)435. Miller, R., Joos, P. and Fainerman, V.B., Adv. Colloid and Interface Sci., 49(1994)249 Miller, T.F. and Meyer, W.C., American Laboratory, February (1984) 91 Mysels, K.J. and Stafford, R.E., Colloids Surfaces, 36(1986)289
67 Mysels, K.J. and Stafford, R.E., Colloids Surfaces, 41 (1989)385 Mysels, K.J., Colloid Surfaces, 43 (1990) 241 Mysels, K.J., Langmuir, 2(1986)428 Mysels, K.J., Langmuir, 5(1989)442 Noskov, B.A., Adv. Colloid and Interface Sci., 69(1996)63 Pugachevich, P.P., Zh. Fiz. Khim., 38(1964) 758 Razouk, R. and Walmsley, D., J. Colloid Interface Sci., 47(1974)515 Rillaerts, E. and Joos, P., J. Phys. Chem., 86(1982)3471. Ross, J.L., Bruce, W.D. and Janna, W.S., Langmuir, 8(1992) 2644 Rusanov, A.I. and Prokhorov, V.A., Interfacial Tensiometry, in Studies of Interface Science, Vol.3, D. M6bius and R. Miller (Editors), Elsevier, Amsterdam, 1996 Simon, M., Ann. Chim. Phys. 32 (1851) 5 Sugden, S., J. Chem. Soc., 121 (1922) 858 Sugden, S., J. Chem. Soc.,125(1924) 27 Van den Bogaert, R. and Joos, P., J. Phys. Chem., 96(1982)3471 Van Hunsel, J. and Joos, P., Colloids Surfaces, 24(1987)139 Volkov, B.N. and Volyak, L.D., Zh. Fiz. Khim., 46(1972)598 Ward, A.F. and Tordai, L., J. Chem. Phys., 14(1946)453. Warren, E.L, Philos. Mag., 4(1927) 358 Woolfrey, S.G., Banzon, G.M. and Groves, M.J., J. Colloid Interface Sci., 112(1986)583
68
Chapter 3
Dynamic interfacial tensiometry of biological liquids obtained from healthy persons All biological liquids of the human organism contain surface active compounds, such as proteins, lipids, and molecules of other nature. These surfactants are characterised by a high adsorption activity at low bulk concentrations which significantly effects equilibrium interfacial properties and the kinetics of physicochemical processes taking place at interfaces (disperse systems of biological liquids, cell membranes). Surfactants may be synthesized endogenously by specific cells or enter the body exogenously through, e.g., the intestine, skin or lungs. Both types of surfactants may undergo various metabolic transformations. Some of the surfactants could be collected in blood others in urine samples. The fact that an appropriate theory was already elaborated, and advanced experimental techniques were available for tensiometric measurements (as discussed in the previous chapters) enabled us to perform systematic experiments with actual human biological liquids. This chapter will describe values of dynamic surface tension characteristics for healthy persons. We will show that dynamic surface tension depend on sex and age. In addition, surface tension characteristics various during pregnancy.
3.1. Dynamic surface tension depend on sex and age All biological liquids of the human organism contain surface active compounds, such as proteins, lipids, and molecules of other nature. These surfactants are characterised by a high adsorption activity at low bulk concentrations which significantly effects equilibrium interfacial properties and the kinetics of physicochemical processes taking place at interfaces (disperse systems of biological liquids, cell membranes). Surfactants may be synthesised endogenously by specific cells or enter the body exogenously through, e.g., the intestine, skin or lungs. Both types of surfactants may undergo various metabolic transformations. Some of the surfactants could be collected in blood others in urine samples.
69 The fact that an appropriate theory was already elaborated, and advanced experimental techniques were available for tensiometric measurements (as discussed in the previous chapters) enabled us to perform systematic experiments with actual h u m a n biological liquids. Table 3.1 summarises averaged values o f dynamic surface tension parameters for serum and urine samples obtained from 80 healthy persons that were between 15 to 65 years old. However, the average data presented b e l o w do not discriminate between persons' sex and age. This will be considered later. Table 3.1. Normal values of dynamic surface tension parameters for serum and urine obtained from 80 healthy volunteers Parameter*
Biological liquid** Serum
Urine
70.0+0.41
71.5 + 0.33
67.7 + 0.35
69.3 + 0.21
60.0 + 0.44
61.8+0.36
X0 [ m N ' m l ' s "I/2]
4.5 + 0.74
4.9+0.65
)~ [ m N / m l . s 1/2]
12.6 + 0.54
13.5 + 0.47
0"1
[mN/m]
0"2 [mN/m]
0" 3
[mN/m]
* Cl = surface tension at t = 0.01 s, 0"2 surface tension at t = 1 s =-
0"3 = 0"~ derived obtained by extrapolation for t ~ oo )~ = ( d 0 " / d t l / Z ) t ~ oo.
9~o = -( d0./dtl/Z)t ~ o,
** Data are given as interval M+3m, with M characterising the average value of a parameter and
m2
the
distribution of this measured value (m2= ~;2/n), where e is the standard deviation, n is the number of volunteers. The interval M+3m corresponds to a probability of 0.9973 that the measured value occurs within the interval [M-3m, M+3m] and may serve as a normal value.
70 For our analysis of dynamic tensiograms obtained from biological liquids we used the following parameters (see last paragraph of Chapter 2 for a detailed explanation): (11 - surface tension at t = 0.01 s, (12 -- surface tension at t = 1 s (13 = (1ooderived obtained by extrapolation for t --~ oo = -(d(1/dtl/2)t -~ o, = (d(1/dtln)t -, oo. In the short time range the surface tensions of serum and urine is by few mN/m lower than that of pure water. The equilibrium surface tensions for both liquids is about 60 mN/m. We have performed an analysis of correlations between various parameters of dynamic surface tensions of biologic liquids taken from healthy persons. The purpose of this studies was twofold. First, high correlation coefficients between certain parameters can be regarded to as an indication for a link between these parameters as they are determined by the same constituents of biologic liquids, or by the same processes which take place therein. If the value of the correlation coefficient between two compared parameters is close to unity, it means that the choice of one of this parameters was inappropriate. On the contrary, low values of correlation coefficients can indicate that these parameters are independent of each other. Second, studies of the correlation between tensiographic parameters under 'standard' conditions, that is, for healthy persons, provides us with an extra tool for the analysis of pathologies, based at the difference of correlation coefficients for certain parameters of dynamic tensiograms. In fact, strong direct correlation (with coefficient r=0.8-0.9) was observed between some parameters of dynamic surface tension for serum and urine (see Fig. 3.1). As expected, a correlation exists in 'neighbouring' surface lifetime ranges ((11- (12, (12- (13). However, the slope L of serum tensiograms does not depend on the other surface tension parameters of this biological liquid, while the L value for urine exhibits a weak positive correlation with the dynamic surface tension crl at t = 0.01 s and (12 at t = 1 s, and a negative correlation with the equilibrium surface tension (13. Dynamic surface tensions of serum depend on its biochemical composition. There exists a strong dependence of (I2 of serum on the concentration of lipids, while ~3 strongly depends on the concentration of proteins (Kazakov
71 et al. 1996a). It can be argued that the excretion of these surfactants via kidneys can lead to the variations in the dynamic surface tension parameters of urine, that is, certain relationships can exist between surface tension parameters o f serum and urine. At the same time, a significant relationship between equilibrium surface tension t~3 and X for urine takes place only with dynamic surface tension parameter or1 of serum (Table 3.2).
a) serum _
0.8
-
0.6O
o r,.)
0.4-
9~
0.2 -
0
0
cj -0.2 -0.4 o1
~2
~3 b) urine
0.8
-
0.6
-
0.4o
o
O
0.2-
-o.2-0.4 -0.6 t~l
t~2
~3
Fig. 3.1. Correlations between surface tension parameters of biological liquids obtained from healthy persons, hatched - cq, black - or2,white - or3
72 Table 3.2. Correlation coefficients between particular surface tension parameters in serum and urine obtained from healthy persons
Serum Urine
0"1
0"2
0"3
0"1
+0.07
+0.23
+0.17
+0.38
0"2
-0.12
-0.13
-0.07
+0.26
0"3
-0.52
-0.36
~
+0.16
+0.53
+0.37
+0.14
-0.06
While surface active constituents of serum are well known and extensively studied, such constituents in urine are rather unknown. Proteinuria in healthy persons is unlikely; however, proteins are mostly responsible for the surface tension of urine even in a healthy person. Normally, slight proteinuria (100-150 mg per day) is attributed to the existence of functional kidney glomerular barriers characterised by selective permittivity with respect to plasma proteins. This permittivity depends on the size, electric charge and configuration of the protein molecules, and also on hydrodynamic factors and the intensity of re-absorption in the tubular apparatus. Among the proteins of unchanged urine, 40% are tissue proteins, secreted by the cells of tubules and mucous coating uropoietic organs, in particular, the Tamm-Horsfall mucoprotein, which possesses a high molecular mass (7000 kDa) and the ability for coagulation within the tubular lumen, forming the matrix of cylinders. Also traces of proteins from the secretion of sexual glands are present in healthy person's urine. All these proteins are likely to determine interfacial tensiometric parameters of urine. The osmolarity of urine probably can affect significantly its dynamic surface tension. Depending on the water/electrolyte balance of the organism, either osmotically concentrated, or hypotonic urine is secreted. The portion of plasmatic secreted proteins in this case is negligibly, and the excretion of osmotically active substances depends on the absolute and relative amounts of soluble electrolytes (sodium, potassium, ammonium). The amount of electrolytes in urine is determined by glomerular filtration, extent of tubular secretion, and reabsorption. These factors, quite naturally, can influence the parameters of dynamic surface tensions of urine.
73 The disbalance in the composition of protein and fat in blood can lead to a hemocoagulation even in healthy persons. This, in turn, can affect the parameters of interfacial tensiometry. For example, if a disbalance in lipid homeostasis exists, then an incorporation of free cholesterol into erythrocyte membranes can happen leading to a change in the cholesterol/phospholipid ratio, accompanied by a transfer of surface active phosphatidyl choline from cells to blood. This surfactant also affects the rheological characteristics of adsorbed layers, increasing its viscosity. Interactions of various metabolites with proteins lead to changes in the molecular structure of proteins, hence they determine changes in their physicochemical characteristics, e.g. viscosity and surface tension. It was mentioned in Chapter 2 that the surface viscosity can affect the results of dynamic interfacial tensiometry in the very short surface lifetime range; that is, surface tension cr~ to some extent reflects also the rheological characteristics of the surface layers. Due to the existence of hydrodynamic effects, a slight influence of the bulk viscosity of liquid also takes place. Table 3.3. Surface tension parameters of serum and urine for healthy persons with respect to sex Biological liquid
Serum
Urine
Parameter*
Male**
Female
or1, mN/m
69.2 + 0.50
70.8 + 0.59
cry, mN/m
67.1 + 0.39
68.3 + 0.54
~2, mN/m
59.3 + 0.18
61.3 • 0.65 w
Lo, m N ' m l ' s ''n
4.9 + 0.90
3.6 + 0.70
~, mN/ml.s 1/2
15.3 + 0.61
8.2 + 0.60 w
G1, mN/m
71.6 + 0.24
71.5 + 0.21
oh, mN/m
69.2 + 0.27
69.3 + 0.32
~3, mN/m
56.6 + 1.81
61.1 + 0.36 w
Lo, mN'm-l's -'n
5.5 + 0.70
5.2 + 0.65
~, mN/m-l.s '~
11.7+0.43
15.2 + 0.54 w
* see comments at table 3.1 **
Sex
see comments at table 3.1
w significantlydifferent between males and females
74 Differences in dynamic interfacial tensiometric parameters exist comparing sex and age (Table 3.3, Figs. 3.2., Fig. 3.3).
76 74
~
7o } 68 ~ 66 64 60 4
~
~
-2
-1
0
1
lg(tef) IS] Fig.3.2. Examples for serum tensiograms obtained from healthy persons of different age: solid thick-male 52 years, solid thin- male 27 years, dashed thick- female 37 years, thin dashed- female 26 years. Equilibrium surface tensions of serum and urine obtained from female are higher than male. The slope of the tensiograms ~. are also different comparing gender: it is higher in serum but lower in urine obtained from males. The relatively high g3 values for female serum can in part be attributed to lower contents of some proteins, lipids and hydrocarbon in their blood. In particular, female serum has lower physiological level of low density and very low density lipoproteins, and a number of ferments (creatine kinase, a-glutamyl transpeptidase, v-glutamate dehydrogenase etc., Kazakov et al. 1996a). In addition, sex related differences exist regarding variations of fractions of phospholipids, cholesterol, triglycerides, free fatty acids, polysaccharides
(galactose,
galactose
amine,
hexose,
fucose),
and
uric acid.
Furthermore, sex related differences exist in the occurrence of protein molecules that contain amino-acids possessing hydrophilic radicals (Grunenberg et al. 1996, Tamagur et al. 1992). One example for a sex related difference in protein concentration is fibronectin. Fibronectin is a high-molecular weight glycoprotein, comprised of two chains which have equal size, linked to each other by disulphide bridges, and contains up to 5% carbohydrates (mannose, galactose,
75 fucose, N-acetylglucosamine, sialic acids). The sources of circulating fibronectin are neutrophiles, macrophages, thrombocytes, fibroblasts, vascular endothelium, hepato- and nephrocytes.
Fibronectin
glycosaminoglycanes,
can be bound
actin,
immune
with
fibrinogen,
complexes
fibrin,
(containing
collagen,
gelatine,
immunoglobulin-G,
immunoglobulin-M, Clq- and C3b-components of complement) (Mosher & Williams 1978 and Baglin et al. 1987). Fibronectin participates in plasma coagulation leading to generation of a fibrin clot. Thus the concentration of fibronectin in serum is 35 to 40% lower than in plasma. The lower the amount of fibronectin in the blood, the higher is the level of circulating fibrinogen/fibrin complexes therein (Vasiliev et al. 1994). The concentration of fibronectin is directly correlated with the concentration of cholesterol, triglycerides and low density lipoproteins. It should be stressed that the content of fibronectin in blood serum for healthy males is much higher than for females (Vasilieva et al. 1991).
75
--
~
70
~
m
""
O.o
i,.,..,.i
65-
60
-2
-1
0
1
lg(tef) [S]
Fig.3.3. Examples for urine tensiograms obtained from healthy persons of different age: solid thick-male 27 years, solid thin - male 19 years, dashed thick- female 48 years, dashed thin- female 30 years. It was shown that an inverse correlation exists between equilibrium surface tension of blood serum for healthy people, and the concentration of such surfactants as cholesterol, triglycerides and fibronectin.
76 Differences between equilibrium surface tension values for persons of different gender may not only due to proteins but can result from different levels of eicosanoides (prostanoides, fatty oxyacids, leukotrienes), and non-lipid (palmitic and hyaluronic acid) or non-protein nitrous components (urea, creatinine, uric acid). Therefore, for males the differences in content and structure of protein and lipid components of urine result in a more pronounced decrease of surface tension. We have found correlations between particular interfacial tensiometric parameters that are sex related. For example, in serum and urine sampled from males the relation between 0"2 and 0"3 is stronger than in samples from females. However, in urine sampled from females equilibrium surface tension parameters depend on ~.. In general, serum and urine sampled from man or women usually show similar sex related correlation links between surface tension parameters. Some of these correlations are stronger than others. Strong correlations exist between X values of urine and 0"1 or 0"2 values of serum in male, see Table 3.4. In females, such correlations were generally not detected, but the relationship between the dynamic surface tension parameters for serum in the short and medium surface lifetime range, and the equilibrium surface tension for urine becomes stronger. For both sexes, the k values of serum are strongly correlated with the 0"1 value of urine. Table 3.4. Correlations between particular surface tension parameters of urine and serum sampled from healthy male or female.
Serum
Sex Urine Male
0"1
0"2
0"3
1"1"
0"1 0"2 0"3
1"1'1" Female
1"1" 1"1'
0"1 0"2 0"3
r162
t - positive correlation; $ - negative correlation; empty- no correlation r < 0.3; one symbol - r < 0.5; two symbols - r = 0.5-0.7; three symbols - r > 0.7
77 In blood of males the concentration of a number of proteins and lipids prevails. Therefore we speculated that a strong positive relation exists between the surface tension parameters of serum and urine in healthy men. However, such a relationship was not observed. The influence of such surfactants on the )~ values of urine (strong positive correlation with ~l and ~2) is difficult to explain. A rather weak, but opposite correlation of equilibrium blood surface tensions for males and females with the dynamic surface tension or1 at t = 0.01 s also remains unexplained. We conclude that dynamic tensiographic parameters depend on some particular, and possibly very complicated combinations of surfactants that are specific constituents of either blood or urine or may exist in both liquids but in different concentrations.. After describing gender related dependencies on dynamic tensiograms, we will now describe age related dependencies. With increasing age, a gradual growth of surface tension of blood, and a gradual decrease of surface tension of urine take place (Figs. 3.4 and 3.5), with most pronounced changes occurring in the very short time ranges.
171513 "
r,r
~
ll 9_
I
I
60
Age [years] Fig. 3.4. Values of ~, (mN.m-1.s 1/2) for serum (II~) and urine ([2) of healthy persons as a function of age.
The increase of
O" 1
values for serum during aging may be in part due to changes of the
biosynthesis and metabolism of proteins and lipids, that leds to changes in the level of surfactants in biological liquids. Examples are insulin and steroid and thyreoid hormones. The production and secretion of which gradually decrease during ageing. In addition, the response
78 of receptors with respect to arginine-vasopressin, adrenaline and thyroxin of target cells in kidney, liver, and the hypothalamo-hypophysiary system deteriorates during ageing (Kazakov et al. 1996a).
a) serum
75 70
m
~' 65
55! 50 +
'
~ t
50
II
68-74
66-71
57-66
10-16
< 20
10
66'70
65-69
58-61
9-15
9
Urine
Males
._
20-35
11
68-74
66-70
59-62
8'14
36-50
10
70-74
67-70
59-65
5-10
> 50
11
70'76
67-72
59-68
4-10
< 20
8
72-73
69-71
61-63
7-12
20-35
10
70-72
68-70
59-62
10-15
36-50
9
70-72
68-70
57-60
10-15
> 50
11
70-73
67-71
56-61
10-16
_
"Females
< 20
10
72-73
69-71
65-68
11-16
20-35
11
70-72
68-70
64-66
14-19
36-50
10
70-72
68-70"
61-65
14-17
70-73
67-71
60-65
12-19
,.
> 50
11
o~ = surface tension at t = 0.01 s (~2 surface tension at t = 1 s or3 = crooderived obtained by extrapolation for t --~ oo L = (dc#dtV2)t ~ o~. Data are given as interval M+3m, with M characterising the average value of a parameter and m2 the dispersion of this measured value (m 2= t~2/n), where e is the standard deviation, n is the number of volunteers. The interval M+3m corresponds to a probability of 0.9973 that the measured value occurs within the interval [M-3m, M+3m] and may serve as a normal value. =
The values for ~, in s e r u m are rather high for children y o u n g e r than 1 year, decrease t o w a r d s the pubertal period, and then sharply increase, attaining its m a x i m u m in the age o f 16 to 20. S u b s e q u e n t l y a gradual decrease o f this p a r a m e t e r takes place. The ~,-values o f urine for juveniles
are
14 m N . m "l .s -1/2.
rather
stable
and
low
(10-12mN-ml.sl/2),
and
increase
with
age
to
81
1715-
I
13-
,
0.7
1"1" $$ 1'1"
$
1'1'
1'1" 1'1'1' 1"1" 1"1" 1"
170 Proteins and lipids, which are strong surface active components, can decrease the surface tension of serum significantly. In our experiments a decrease in surface tension of serum with increased concentrations of these compounds was observed only for short and medium surface lifetimes (for t = 0.01 s and t = 1 s), while their effect on the equilibrium surface tension was rather unexpected. Alterations of the physicochemical properties of proteins for patients with diabetic nephropathy can be hypothetically ascribed to the glycosilation process, that is, the non-fermental attachment of glucose to the amino groups of proteins. The glycosilation is a non-specific reaction, which involves proteins of serum, basal membranes, erythrocyte membranes etc. The flexibility of blood cell membranes depends on the existence of a lipid bilayer and proteins, capable of being glycosilated. Violations of the carbohydrate exchange initiate disturbances in the kidney microvessels (Galenok & Chanykina 1991). Urea, creatinine, uric acid and other low molecular compounds affect the structure of proteins, making the amino groups less accessible for linking with glucose; this leads to violations in the protein glycosilation processes (Lebedeva 1996, Barbagallo et al. 1993). We believe that this fact can explain the decrease of equilibrium surface tensions of blood for patients suffering from diabetic nephropathy with chronic renal insufficiency (g3 is negatively correlated to the nitrous non-proteinic constituents of blood serum). For diabetic nephropathy, pronounced dependencies exist between surface tensiometric parameters and the amounts of immunoglobulins and circulating immune complexes present in blood. It is known that serum of patients suffering from diabetes mellitus of type I (insulindependent) and II (insulin-independent) exhibits an increase in the immunoglobulin level. Usually the concentration of immunoglobulins-G becomes higher. It was argued, however (Saltykov et al.
1990) that a concentration
increase
of immunoglobulins-A
and
immunoglobulins-M also takes place. Our data show that hyperimmunoglobulinemia results in a concentration
increase
for all
classes
of such proteins,
where
the
increased
immunoglobulin-G level is most pronounced. However, their concentration becomes significantly lower with the development of a nephrotic syndrome. Patients with diabetic nephropathy exhibit large amounts of circulating immune complexes able to fix the C3-component of the complement and to incorporate insulin. Close correlations were found between the existence of a renal microangiopathy and the presence of circulating immune complexes containing immunoglobulins-A1 and immunoglobulins-A2.
171 The proteinuria is the most early symptom of kidney affection for diabetes mellitus, while structural lesions of the kidney tissue arises much earlier as proteinuria becomes recognisable. The earliest preclinical signs of diabetic nephropathy are the microalbinuria (the excretion of albumins below the threshold of common methods used for the detection of proteinuria, i.e. 30-300 ~tg per day) and high urinal concentrations of the specific ferments localised in the epithelium of kidney proximal tubules. While the mechanisms of glomerular and tubular disturbances for diabetes mellitus are different, a direct correlation between microalbuminuria and the activity of acetyl-[3-D-glucosamine peptidase and alanineamine peptidase in urine exists (Jung et al. 1989), suggesting that a high excretion of these enzymes is a marker of diabetic nephropathy. The damage of the tubular epithelium leads to urinal excretion of ferments specific to the membranes of proximal tubule brush border (alkaline phosphatase, ,/-glutamyl transferase), cytoplasm (LDG) and lysosomic ~-glucuronidase (Dedov et al. 1989). The enhanced urinal excretion of low molecular [32-microglobulins indicates a change in the tubular reabsorption process. Therefore, the increase in urinal excretion of surfactants, such as albumins, ferments and [32-microglobulins, can lead to varied dynamic surface tensions even at a preclinical stage of diabetic nephropathy. For pronounced glomerulosclerosis, the dynamic surface tensiometry parameters of urine will exhibit most appreciable variations. For diabetes mellitus, activated T-lymphocytes become capable of producing a factor which enhances the penetrability of vessels and the synthesis of endoglycosidase - the ferment for the degradation of heparane sulphate of protein glycanes of the subendothelial matrix and basal glomerular membrane. Heparane sulphate determines the negative charge of the glomerule filtration barrier, and acts as a physiological anticoagulant. Therefore, a lack of negative charge of the glomerular filter, a decreased local anticoagulant potential, and extended penetrability of the vessels (caused by products of activated lymphocytes) promote the enhanced excretion of negatively charged albumins (Salozhin et al. 1991). An interrelation exists between albuminuria and the concentration of lipoproteins in blood serum (Muchin et al. 1990, Schnack et al. 1994). Each diabetes mellitus patient without any clinical-laboratory symptoms of kidney damage displays a change in the glomerular filtration, while for one patient out of five an affection of the proximal tubules was found. In 75% of all these cases a hidden proteinuria was found by
172 special tests. The presence of proteins with an electrophoretic mobility characteristic to prealbumin, albumin and post-albumin in urine was found for 6% of patients suffering from diabetes mellitus of type I, while for type II this portion increased to 41%. For transferrin and haptoglobulin these percentages are 38% and 59%, respectively. The presence of uromucoids in the region of immunoglobulins and tx2-microglobulin was detected for 57% of patients with diabetes mellitus type I (insulin-independent) only. The increase of urinal excretion of surfactants filtered and secreted by kidneys for diabetes mellitus type I leads to decreased o2 values. The pronounced proteinuria for patients with nephrotic syndrome leads to strongly decreased surface tensions of urine (cf. Fig. 4.32). Hampered reabsorption processes in kidney tubules for chronic renal insufficiency result in more pronounced shifts of the dynamic tensiograms at medium surface lifetimes (cf. Fig. 4.31). The values of 0.1 for urine correlate directly with the parameters 0.2 and 0.3 of this biological liquid. The correlation between the parameters in the medium and large time range is still more significant. There is a strong negative dependence of the ~ values for urine on the equilibrium surface tension. Thus, all the correlations just mentioned indicate that the variation of dynamic tensiometry parameters for this type of the disease are caused by single reason. Interrelations between the tensiographic parameters of different biological liquids also exist. In particular, the ~. value of serum for diabetic nephropathy is related to 0.2 and 0.3 of urine, and 0.3 of urine correlates with the parameter 0.2 of serum (cf. Fig. 4.30b). The dynamic surface tensiometry parameters for urine exhibit inverse correlations with the duration of diabetes mellitus, but do not depend on the glucosuria level. However, there are weak negative dependencies of 0.1 and 0.3 for urine on the glucose concentration in blood (Fig. 4.33). A correlation between the extent of the albuminuria and the 0.a-value for serum is observed as well. The level of albuminemia should determine the values of equilibrium surface tension for serum; therefore for patients with diabetic nephropathy, the concentrations of albumin in various biological liquids will affect significantly the value of c3 in serum. In this connection, both the direct correlation between the albumin concentration in serum and equilibrium surface tension of serum, and the inverse correlation between the concentration of albumin in urine and 0"3 of
serum remains unclear. First, increased concentrations of albumin cause a surface tension
fall of the model solutions; second, the albumin contents in blood decreases with increased
173
proteinuria. Hence the two biological liquids exhibit opposite dependencies with respect to the albumin concentrations. These anomalies of surface tension are possibly related to the conformations of proteins caused by the interaction with low molecular nitrous compounds, and a variation in their adsorption properties caused by the glycosilation processes. Senna
Urine
0.8 0.6
-
0.4o
O
0.2-
I
-0.2-0.4 -0.6 al
a2
a3
k
al
a2
a3
L
Fig. 4.33. Correlation coefficients between surface tension parameters measured in biological liquids obtained from patients with diabetic glomerulosclerosis and the level of glycemia.
0,2
-
0,1 ~
0
~
-0,1 -
O
0,2
=
0,3
7/.,_-s $22
:::" "":" .... :" ..... "::" ............ : ::": """ ":":": :"" ..... ""' ..... : :" ::::::: : ......
0,4
.............
L!.;.
"~
-05
7_72 S7_,,ii 7"",.........i $2 .. ,,.;TZ ,',"..'.'?Z 2"Z"S ZZ2 ?7.,221 ,SZ
75.S 77,_,S
-~
-0,6 -
~ i -0,7 -0,8 -
P
A
F
MG
Fig. 4.34. Correlation coefficients between surface tension parameters measured in urine obtained from patients with diabetic glomerulosclerosis and the concentration of proteins in urine. Surface tension parameters are o~ - hatched, tr2- black, a3- white and ~ -grey. Measured proteins are albumin - A, fibronectin- F, 132-microglobulin - MG, total protein- P.
174 There exists quite expectedly a negative correlation between urine tensiographic parameters and the concentrations of various proteins (cf. Fig. 4.34). This is primarily true for the parameters of surface tensiograms in the short time range. Both fibronectin and 132-microglobulin affect the Z, value of urine. As chronic pyelonephritis often accompanies diabetes mellitus in general and diabetic nephropathy in particular, the results obtained by surface tensiometry of biological liquids for female patients possessing this pathology have been analysed carefully in that respect. It was mentioned above that for female patients suffering from primary pyelonephritis, decreases in o2 and
03
for serum, o2 and ~, for urine, and increased values of ~, for serum were observed.
One should naturally expect equivalent variations of surface tension parameters for diabetic nephropathy accompanied by chronic pyelonephritis. On the contrary, it was found that this combination results in an increase (not decrease) of o2 of urine and of the equilibrium surface tensions for both the biological liquids (cf. Fig. 4.35). The only tensiographic parameter whose increase was reliably stated for chronic pyelonephritis, is ~ for serum.
Serum
4q 24 0-2
-
-4
-
Urine
-6 -8 10 -12 i -14 j 16 18 ol
o2
o3
ol
o2
o3
Fig. 4.35. Changes in surface tension parameters measured in biological liquids obtained from patients with diabetic nephropathy. Black columns- without accompanyingpyelonephritis, white columns- with accompanyingpyelonephritis. Changes are given in % comparedto correspondinghealthy controls.
175 To summarise, the diabetic nephropathy combined with pyelonephritis results in quite unusual variations of the dynamic surface tensiometric parameters, characteristic neither for primary pyelonephritis, nor for isolated diabetic glomerulosclerosis. Therefore, a future task in these studies should be to compare the characteristics of surface tensions between the patients who do not suffer from the renal syndrome and possess quite similar parameters of the renal function, similar arterial blood pressure, disease duration, etc. It would be interesting to study the surface tension of biological liquids for patients suffering from diabetes mellitus causing no renal lesions, as compared with pyelonephritis not accompanied by diabetic nephropathy. At the present stage it can be argued that for the cases of diabetic nephropathy, the dynamic surface tensiometric parameters can be regarded as rather informative criteria for differential diagnosis, which enable one to predict further developments of the pathologic process. We believe that dynamic surface tension studies of serum and urine will become a reliable auxiliary method for monitoring various treatments. 4.4. Other renal diseases
Other renal disease that were studied using dynamic surface tension analysis of biological fluids include podagric nephropathy (40 patients), kidney amyloidosis (21 patients), kidney sarcoidosis (19), hypertension disease accompanied by nephrosclerosis (18), myelomic nephropathy (18). All these diseases either are kidney specific or affect kidneys secondarily. Among other renal diseases, the amyloidosis is remarkable for the hyperproduction and the decrease in the catabolism of free light chains, and the structural components of immunoglobulins (Resnikov et al. 1996) having surface activity. For the primary variant of this disease, the amyloid fibrillae consist of polypeptide fragments of normal serum proteins and light chains of immtmoglobulins or their fragments. For the secondary variant, the amyloid protein differs in its amino acid sequence, and is represented by the protein AA (molecular mass ca. 9 kDa), which forms from the serum albumin SAA. For the hereditary form, the amyloid fibrillae were identified as the prealbumins (with a molecular mass in the interval of 8 kDa to 40 kDa). The P-component of any amyloid deposition corresponds to the blood serum glycoprotein (molecular mass 23 kDa), and contains large levels of glutaminic and aspartic acids, glycine, leucine, and, in a lower extent tryptophane.
176 The protein SAA is synthesised by hepatocytes, and becomes present in blood in significant amounts as a result of prolonged antigenic stimulation. In the blood circulation, this protein is closely related to the high density lipoprotein fraction, forming complexes with them (with molecular masses around 180 kDa). For all types of the disease, the amyloid fibrillae are of pronounced polyanionic character, and therefore bind other proteins (Bannikova 1987). The specific composition of blood proteins for kidney amyloidosis should affect the state of dynamic surface tensions of biological liquids - not only of serum, but also of urine, because pathologic proteins are filtrated in the kidney glomeruli. Surface tensiometry of biological liquids has been performed for patients with secondary kidney amyloidosis caused by rheumatoid arthritis (the diagnosis was confirmed by biopsy). Decreased surface tension of serum in the short and medium surface lifetime range, and a decrease in the ~. values were observed (cf. Fig. 4.36).
75
....
70
.o
.............. ......
~.............
~ 1 7 6O~176176 176 ~176176 ~ ~ o~176 ~ ~ o~ ~ ~ ~176
t_.__a
~ ~ ~176 ~176
[
60
55
t
-2
. . . .
-1
t
b
t
0 lg(tef) [S]
1
2
Fig. 4.36. Example of serum tensiogramobtained from patient (female, age 58) with rheumatoid arthritis accompanied by secondary kidney amyloidosis,nephrotic syndrome,chronic renal insufficiencyof 2nd stage, dotted line correspond to average value for healthy females of the same age. The urine of such patients is characterised by high
(Yl
and
03
values, with a significant decrease
in the ~, values. It should be stressed that for all patients screened, the nephrotic syndrome and normal renal ftmctions were diagnosed.
177 When morphologic studies of the nephrobioptates are not available, clinicians encounter severe difficulties in the differential diagnosis of kidney amyloidosis and chronic glomerulonephritis. It should be recalled in this connection that the nephrotic syndrome for chronic glomerulonephritis is characterised by a significant increase in ~, for serum and decreased ~2 and ~3 values of urine, while for acute glomerulonephritis an increase in L of serum and the Crl values of urine is observed. Therefore the results of dynamic surface tensiometry for biological liquids enables one to distinguish rather reliably between glomerulonephrites and kidney amyloidosis at the nephrotic stage of the pathologic process, which is of a significant practical value. One approach to the study of the intensity of cellular receptors metabolism is the determination of R-protein concentrations in biological liquids. These are products of the catabolic decomposition of external parts separated from the cells. The serum R-proteins are capable of binding ligands of catalytic activity similar to that of superoxyde dismutases, and can circulate either in a free state, or in complexes formed with immunoglobulin-G. For kidney amyloidosis, the level of R-proteins in blood increases as the result of the peculiar (~desquamation>> of cells; this leads to a distortion of their response to various stimuli (Kozlovskaya et al. 1992). The concentration of R-proteins, rather expectedly, displays an inverse correlation with surface tension parameters of serum in the short and medium adsorption time range. For patients suffering from kidney amyloidosis, the contents of fibronectin in serum is somewhat higher than that characteristic of chronic glomerulonephritis. The level of fibronectinuria increases sharply, especially for patients with a nephrotic syndrome. The urinal excretion of this high molecular plasmatic glycoprotein is enhanced due to the increase in the penetrability of the vessels, the glomerular filtration of the glycoprotein, and the intensified metabolism of glomerular capillary basal membranes (Karryjeva et al. 1992, Westermark et al. 1991). The dynamic surface tensions of urine in the short time range exhibits direct correlations with the concentration of fibronectin in urine for kidney amyloidosis accompanied by the nephrotic syndrome. The monoclonal free light chains take their part in the pathogenesis, not for the amyloidosis only, but also for the myelomic disease. They possess a nephrotoxic effect, and determine the prognosis of the disease evolution. Low molecular free light chains are easily filtered and
178 almost completely reabsorbed in the kidneys. However, when the number of functioning nephrones decreases and, therefore, the glomerular filtration becomes less intensive, then these free light chains cannot arrive in the region of their catabolism. This results in a decrease of their excretion, and an increase of their contents in blood. A reliable marker for highly differentiated B-cell tumours, one example of which is the multiplex myeloma, is the production of monoclonal immunoglobulins and/or free light chains -
Bens Jones protein. While the diagnostics capability for serum monoclonal immunoglobulins
is by no means absolute, the presence of free light chains in urine can be regarded as evidence for the tumoral nature of a process. In spite on the fact that K-chains are much more frequently incorporated into immunoglobulins-G, there are Z,-chains which prevail in free light chains (because they are less capable of binding to immunoglobulins-G). The same difference exists between the polyclonal and monoclonal free light chains: in monoclonal free light chains the occurrence of K-chains is twice as high as that of ~-chains. In the pre-clinical stage of myelomic nephropathy, moderate effects of nephrothelium albuminous degeneration can be detected, while no changes in glomeruli and interstice are as yet present. Then a pronounced granular hyaline and hydropic degeneration develops, along with a moderate atrophy of tubular epithelium and eosinophilic cylinders in their lumina. An ectasia of stroma takes place with separate focuses of sclerosis. Further stages of the development of myelomic disease are characterised by the thickening of the glomerular capillary basal membranes, an increase of the mesangium, and an extensive process of glomeruli elimination, controlled by the periglomerular (or, less frequently, capillary collapse) sclerotic mechanism. Finally, the nephrocalcinosis becomes evident (Sidorova et al. 1988, Ivanyi 1993, Mundy 1990). One could expect similarities between the parameters of surface tension of serum for myelomic disease and those for kidney amyloidosis. However, the multiplex myeloma is characterised by decreased surface tensiometric parameters of seruna in the medium and long time range, with a significant increase of ~,. It has to be noted that the myelomic disease is accompanied by hyperproteinemia and more pronounced ~-globulinemia, relatively low contents of cholesterol and triglycerides in blood, even for cases of myelomic nephropathy with nephrotic syndrome. Typical for such patients is the increase of crl and X values of serum. The development of amyloidosis for myelomic disease leads to an increase in the or3 and ~ values of urine.
179 The dynamic surface tensiometric parameters in the medium surface lifetime range (t = 1 s) directly correlate with the total concentration of proteins and the level of immunoglobulins M and G in blood. The parameter 0-1 for urine depends inversely on total proteinuria (not albuminuria solely), while
0"3
inversely depends on the fibronectinuria.
While the hypercalciemia, hyperuricemia and high blood viscosity contribute significantly to the development of nephropathy for multiplex myeloma, the main role is believed to be played by the renal excretion of anomalous immunoglobulins (proteinuria of repletion) whose accumulation in the interstice, glomerular and tubular basal membrane leads to a damage of the nephrothelium and locking of tubular lumen. The plasmapheretic treatment of such patients makes it possible to remove large masses of pathologic proteins, to decrease plasma viscosity and the oncotic pressure, to improve the rheologic properties of blood and the performance of microcirculation processes in kidneys (Abdulkadyrov et al. 1991, Abdulkadyrov and Bessemeltsev 1992, Reinhart et al. 1992). Reciprocal variations of blood macroproteins and albumins, rather moderate for the plasmapheresis,
become
more
significant
for
the
cytapheresis
and,
especially,
plasmacytapheresis. At the beginning of the second stage the level of macroproteins somewhat exceeds its initial value, while the concentrations of albumins and globulins remain lower than their initial values. As the relative contents of proteinic fractions in blood is rather inertial, the level of macroproteins can be regarded as merely the general indicator of the treatment efficiency. Repeated procedures lead to the increase in the total amount of eliminated proteins and macroproteins. One can presume that at least two mechanisms for the compensation of the blood proteinic system exist in respect to a plasmapheresis treatment, a fast and a slow mechanism, respectively. The fast mechanism becomes active at the commencement of the procedure, and controls efficiently the proteinemia parameters during tens of minutes, while the slow mechanism remains effective after many hours. The fast mechanism is supported preferentially due to the penetrability, deposition and re-deposition of proteins and liquid constituents of blood. The crucial role in the performance of the second mechanism is played by processes of biosynthesis of plasma proteins.
180 Variations in the total concentration of proteins, and the decrease of the albumin/globulin factor for the myelomic disease, lead to the increase of the plasma colloid-osmotic pressure. The plasmapheresis process leads to the elimination of globulin fractions of the proteins, to a decrease of the levels of proteinemia, circulating immune complexes, and blood viscosity. The rheological properties are improved due to the normalisation of the plasma colloid-osmotic pressure (osmolarity). The application of a treatment makes it possible to eliminate the paraproteins, to improve the coagulation state of the blood and the drainage function of the tissue.
410 . . 390 ~
m m
370
m
--
m
m
m
350 O .~
330-
_~
310
0
~ 9 r~
O""""M+3m
<M-3m
0"1
76.7
18.6
0"2
62.8
14.0
0"3
67.4
9.3
7.0
53.5
0"1
41.9
16.3
0"2
51.6
27.9
0"3
53.5
37.2
9.3
74.4
Table 5.9. Correlation coefficients between surface tension parameters measured in biological liquids that were obtained from patients with rheumatoid arthritis.
Biological liquid
Urine
Synovial fluid
Surface
tension
Serum
parameter
0"1
0"2
0-3
0"1
+0.06
+0.12
+0.17
+0.03
0"2
-0.03
+0.16
+0.15
+0.17
0"3
+0.02
+0.29
+0.26
+0.29
+0.01
-0.22
-0.24
-0.20
0"1
+0.73
-0.25
+0.62
-0.70
0"2
+0.85
+0.02
+0.81
-0.88
0"3
+0.77
-0.18
+0.69
-0.73
+0.46
+0.59
+0.54
-0.61
219
a) serum 0.9
-
0.8 .~ 0.7 -:--:-..-:.:-
~ o
o
C
-:-:-:-:-:-:-.. ....... :-.... .... :..:...:.-. ". .:.'.:.' .: .2 2 " : ' : ..... ..-. :-...:-:.-. ". .:.2. .2. 2. " : 2 2 ~:..-.-...: ..:..-.-..: ~-.-.-.-.:-. -. .: .-.:.-.:.- : - - : - : .".:.' .:.' .: .' . ' 2 " : ' : -:-:-:-:-:-:-: ....... ..-...-.- ..-. .: .-.:.:.:.T. : ..-...-.-..: :-.......... -...... .... :..:........ ............ ........ -. .. . . . . ..-. ...........-. .. . . . . ..-.
0.6 0.50.4-
0.3 oo
:. :. .- .:.-.-.:. -. :. .- .f : :-.. .....
0.2-
"-.222222 ...... -.
:....-.......
0.1
iiiiiiiii ?:-55555:
I
I
o'1
o2
0"3
b) synovial fluid _
0.8 0.6 0.4 0~
o
0.20 -0.2 -0.4-
o
-0.6 -0.8 -1crl
or2
o3
Fig. 5.14. Correlations between various dynamic surface tension parameters of biological liquids obtained from patients with rheumatoid arthritis. Hatched - o 1, black -o2, white - or3, grey - X.
Of some interest are the data concerning the variations in the tensiograms of serum for patients with the seropositive version of the disease, when the rheumatoid factor in the serum leads to a sharp increase of the surface tension (cf. Figs. 5.16 and 5.17). One can presume therefore that the concentration of immunoglobulins, which contributes to the formation of the rheumatoid factor, can affect the surface tension of serum for rheumatoid arthritis.
220
Activity degree
Stage
10
~
I
JI
I
,,'.,, ~,.~
"~ -20 -25 t -30 t -35 -40 ~
I
II
III
I
II
III
IV
Fig. 5.15. Changes in surface tension parameters measured in serum obtained from patients with rheumatoid arthritis and various activity degrees indicated as I, II, III and various stages of the disease indicated as I, II, III, IV. Changes are given in % compared to corresponding healthy controls. Hatched - a~, black - a2, white - a3, grey - ~,.
25201510-
t_____a
5
..o
0 -~
;>
! !
-5 -10 -15 -20 -25 al
a2
a3
~,
Fig. 5.16. Changes in surface tension parameters measured in serum obtained from patients with rheumatoid arthritis for various serologic activities of the disease; black - seronegative, white - seropositive. Changes are given in % compared to corresponding healthy controls.
221
75
-............
_....
o. . . . . . . . . . . . . . .
7 0 - -
~ .............
65
~ ~ ~
E 60 Z ~-~55 50 45 40 -2
I
I
I
-1
0
1
lg(tef) Is]
Fig. 5.17.Examples for serum tensiograms obtained from patients with rheumatoid arthritis, one with seropositive version (female, age 69, thin line), one with seronegative version (female, age52, thick line); dotted curves correspond to average values for healthy females of corresponding age. In some cases, common laboratory methods fail to detect the rheumatoid factor. First of all, for 1/5 of all patients, the immunoglobulins-G are blocked by the immunoglobulin-M-rheumatoid factor; secondly, the immunoglobulin-G-rheumatoid factor, which cannot be registered by known agglutination tests, can be present instead of the immunoglobulin-M-rheumatoid factor, recognised as "seronegative" cases (Aho 1986). It is quite possible that more precise randomisation of the patients with respect to serologic versions of the disease (we have used the Waaler-Rose and latex-test) could affect the mean surface tension parameters of serum. We believe, however, that the variations detected would in any case display at least pronounced trends. It should be noted that the dynamic surface tension of serum increases with the duration of rheumatoid arthritis (cf. Fig. 5.18), becoming apparent only after 7 years with a twofold increase of the parameters after about 15 years. We believe this phenomenon can be explained either by the formation of some new substances in blood, or by the fact that substances already existing can acquire unusual surface active or surface inactive npon~pxt~cy (e.g. due to the effect of medication).
222
1210~
o .~
8._o
64-
21 1
1
3
1
5
I
7
I
9
I
11
I
13
15
Duration [years]
Fig. 5.18. Changes of surface tension parameters measured in serum obtained from patients with rheumatoid arthritis as a function of the disease duration. Changes are given in % compared to corresponding healthy controls. (~) - ol, (m) _ o2, (A) - o3. It was already mentioned in the previous chapter that variations in surface tensiograms of urine appear during the development of a secondary kidney amyloidosis for patients suffering from rheumatoid arthritis. At the same time it should be noted that even if no clinical-laboratory symptoms of any nephropathy exist, then the ~, value of urine decreases. These deviations from characteristic values of healthy subjects were found in 3/4 of all cases of rheumatoid arthritis studied (cf. Table 5.8). It is well known that kidney lesion can result from rheumatoid arthritis. In addition to the formation of amyloidosis, glomerulonephritis and interstitial nephritis often occur, the development of which can be latent, without apparent symptoms. Morphological studies show the thickening and doubling of the tubular basal membrane, albuminous degeneration and focal atrophy
of
the
epithelium
of
convoluted
tubules,
stroma
oedema
and
sclerosis,
lymphohistiocytic infiltration, hyalinosis of arterioles, segmental ectasis and sclerosis of glomerular mesangium, focal proliferation of mesangial cells, sometimes accompanied by the thickening of the capillary basal glomerular membrane, external capsules, and sclerosis of vascular ansae (Krel' et al. 1990). The detection of mesangioproliferative glomerulonephritis
223 using kidney biopsy is possible even if the urine analysis shows no changes, and the symptoms of the uric syndrome do not necessarily correspond to the extent of kidney pathology. While the equilibrium surface tension and L of serum for patients who do not suffer from cardiac or hepatic pathology are virtually equal to the reference values of respective characteristic parameters in healthy persons, the existence of cardiac or hepatic pathology is accompanied by an increase of 0-3 and a decrease of L. Note that the cardiac degradation does not result in any changes of 0-1 and 0"2 for serum, while the hepatic pathology leads to an increase of these surface tensiographic parameters. Table 5.10 demonstrates the correlations between surface tension parameters of serum and concentrations of some proteins and lipids. The results obtained in these studies are quite complex, therefore some additional comments are to be made. In fact, a direct correlation has been expected between the dynamic surface tension and the contents of these surfactants in blood, because the surface tensiographic parameters for serum often exhibit an increase when the activity of the pathologic process increases. In turn, the activity of rheumatoid arthritis positively correlates with the levels of immunoglobulins and 132-microglobulin. It is quite possible that the state of surface tension is determined also by other constituents of serum (including those of non-protein and non-lipid nature). The answer to this question is as yet unknown,
The synovial fluid is the most available indicative medium regarding the character of the articulatory lesion. It should be kept in mind that 0-1 and ~, for synovial fluid are lower than those of blood. At the same time, synovial fluid of patients with rheumatoid arthritis possess large concentrations of immunoglobulins-G, immunoglobulins-M and immune complexes which contain large quantities of immunoglobulins G, M and A (Shine et al. 1991). The activity of acidic phosphatase exceeds 8 times that characteristic for patients suffering from posttraumatic arthritis, while the acetyl-13-D-glucosaminepeptidase activity is three times as high as that for posttraumatic arthritis, and two times higher than that for patients suffering from Reiter's disease. The level of 132-microglobulin is twice as high as the contents of this protein in blood, and correlates with the total amount of protein in synovial fluid. During the phagocytosis of immune complexes by neutrophiles and mononuclear phagocytes, lysosomic ferments are released to the extracellular medium (Henderson & Pettinher 1985).
224 Table 5.10 Correlation coefficients between surface tension parameters measured in serum obtained from patients with rheumatoid arthritis and serum components.
Serum component 0"1
0"2
0'3
$$
$$
$$
$$$
$
$ $
$$
$$
$$$
$$
Total protein Albumin ot~-globulin fraction a2-globulin fraction 13-globulin fraction T-globulin fraction Immunoglobulin-G Immunoglobulin-A
$$
$$
Immunoglobulin-M 132-microglobulin Fibrinogen Fibronectin
i"
Circulating immune complexes Total cholesterol
$
or-cholesterol Triglycerides High density lipoprotein fraction Low density lipoprotein fraction Very low density lipoprotein fraction
$
$
1" p o s i t i v e c o r r e l a t i o n ; ,1, n e g a t i v e c o r r e l a t i o n ; e m p t y - no c o r r e l a t i o n r 0.7
Synovial fluid of patients with rheumatoid arthritis contains large immunoglobulin-G complexes, which can react with the immunoglobulin-M-rheumatoid factor forming very large, stable and insoluble intercross-reacting compounds. Also specific antibodies directed against immunoglobulins are found, which cannot be regarded as rheumatoid factors, because they
225 react with other regions of the immunoglobulin-G molecule or with immunoglobulins belonging to other classes. Immunoglobulin-G-rheumatoid factor is most often found in biological liquids that contain many
immunoglobulin-G-immune
complexes.
Even
at
low
concentrations
of
the
immunoglobulin-G-rheumatoid factor, immunoglobulins-G dimerise due to self-association. The significant level of immunoglobulins-G in blood results in an excess of antigen and prevents from the formation of increased size of immune complexes, therefore these complexes are small and usually incapable of binding to complement. There is a significant amount of immunoglobulins-G-rheumatoid factor in the joint cavity, which reacts with normal immunoglobulins-G with the formation of immune complexes that undergo partial aggregation. The attachment of immunoglobulins-G-rheumatoid factor and complement to such megacomplexes leads to a further increase of their size. The molecules of normal immunoglobulin-G and immunoglobulin-G-rheumatoid factor possess two antigen determinants in the region of the Fc-fragment which bind the immunoglobulins-G-rheumatoid factor. This leads to a rotation and steric blocking of the second determinant of unchanged immunoglobulin-G, distorting the position of the Fab-fragment. Thus the excess of normal immunoglobulins-G prevents a further polymerisation of immunoglobulin-G-rheumatoid factor. Their dimers cannot block the second determinant; therefore large polymers are formed (Munthe & Egeland 1984). Such remarkable differences in the composition of immune complexes in blood and synovial fluid is responsible for the diversity in the variations of surface tension between these biological liquids. Morphological studies of the synovial membrane, even for a prolonged course of seronegative rheumatoid arthritis, do not display the classic appearance of rheumatoid synovitis. The proliferative reaction of synovial cell is observed with the formation of multilayer strata, the hypertrophy of the endothelium of venulae and capillaries of microcirculatory bed with a deterioration of the penetrability of vessels and the deposition of fibrinous masses. No antibodies are locally synthesised or lymphoid-plasmacellar follicles formed (Ivanova et al. 1986, Busso et al. 1998). One of the most significant mechanism, which cause a distortion in the metabolism of the bone/cartilage matrix in rheumatoid arthritis is the pathology of the metabolism of tissue
226 (cartilage) proteoglycans. Both the concentration and the qualitative composition of proteoglycans and glycosaminoglycanes affect the morphological and functional state of the connective tissue. Certain dependencies were observed in the variation of the contents and qualitative composition of glycosaminoglycanes in synovial fluid for various degrees of synovitis. A direct correlation between the gravity of a local inflammation process and the amount of sulphated glycosaminoglycanes exists, and an inverse correlation with nonsulphated (by hyaluronic acid) glycosaminoglycanes (Astachova et al. 1989, Silbermann et al. 1990, Bensouyad et al. 1990). Hyaluronic acid, which is the main glycosaminoglycane among those constituting the synovial fluid, determines its viscosity. For articular diseases the viscosity of synovial fluid decreases, which can be explained by the depolymerisation of hyaluronic acid or the formation of lowpolymeric hyaluronates due to demage of the synthesis process. This depolymerisation is caused by the action of a number of lysosomic ferments (e.g., [3-glucuronidase) and peroxide radicals. The increased contents of chondroitynsulphates in synovial fluid correlates with the gravity of the articular inflammation, and is determined by a destruction of tissue (cartilage) proteoglycans. These molecules possess a unique property, the ability to form large aggregates, comprised of proteoglycane sub-units, hyaluronic acid and connecting proteins. This protein stabilises the proteoglycane-hyaluronic complex, preventing its dissociation. The chondrocytes which produce glycosaminoglycanes and are influenced by lysosomic ferments, perform the synthesis of anomalous proteoglycans, incapable of aggregation; therefore these chondrocytes are more vulnerable to the action of hydrolases. The discussion above can also explain differences in surface tension between blood serum and synovial fluid for patients with rheumatoid arthritis. Rheumatoid arthritis is accompanied by a decreased activity of the fibrinolytic system in synovial fluid. Increased amounts of fibrin degradation products were found, which correlate with the concentration of coagulation factor XI and XII. The deficiency in the amount of plasminogen activators is caused by a decrease in their synthesis by the articulation synovial membrane. At the same time, the concentration of plasminogen activation inhibitors is increased, which suppresses the fibrinolysis. Eventually, the contents of fibrinogen and fibrin
227 in synovial fluid increases. The deposition of fibrin at the synovial membrane hampers the exchange between the articulation liquid and the cartilage (Murav'iev et al. 1989, Clemmensen et al. 1983). For rheumatoid arthritis an increase of the fibronectin concentration in synovial fluid was observed (Vasil'eva et al. 1991, Scott et al. 1981). The analysis of the composition of polyethylene glycolic precipitates in articulation liquid has shown that fibronectin as their integral constituent, can be related to immunoglobulins, rheumatoid factor and the components of the complement. There are some differences between fibronectin molecules in synovial fluid and those in blood serum, therefore the contents of proteins in the immune complexes of these biological liquids is different. For patients suffering from rheumatoid arthritis, a degradation of fibronectin molecules take place and complexes with other proteins are formed. It should be stressed that for arthritis with other etiology, the contents of fibronectin in synovial fluid virtually does not change. For healthy subjects fibronectin is the only protein, which has a concentration in the articular liquid similar to that in serum, while the amount of other synovial proteins (which originate from blood) is significantly lower. This indicates an important contribution of local synthesis of fibronectin by synovial cells and/or cells which are present in the synovial cavities. The variations in the amount of synovial fibronectin are accompanied by a development of its physicochemical inhomogeneity. The pathological forms of the protein and the derivatives of fibronectin can differ significantly from the native forms in their bonding with gelatine, which is the product of collagen denaturation (Abdullin et al. 1988). A direct correlation link exists between the gelatine-bonding ability of fibronectin in synovial fluid and the gravity of the rheumatoid arthritis. The malfunction of the fibrinolytic system Of synovial fluid for patients with rheumatoid arthritis controls in many ways the dynamic surface tensiometric parameters. In the short surface lifetime the surface tensions of serum exhibit some correlations with the concentrations of fibrinogen and fibronectin (cf. Table 5.10). For synovial fluid, this dependence becomes more pronounced, and a correlation to the value of tensiographic quantity ~. becomes apparent, having however negative correlation coefficients. Therefore it can be argued that other factors are responsible for the differences in surface tension of serum and synovial fluid.
228 5.6. Reiter's disease
Reiter's disease is characterised by an increase of and an increase in cl and
(Y3
13'1 and
decrease of ~, for serum tensiograms,
for urine. Surface tensiographic parameters of synovial fluid are
significantly lower than corresponding serum parameters, see Fig. 5.19. Such finding could be explained by the differences in the contents of surface active and inactive substances in these biological liquids.
In particular,
synovial fluid contains lower amounts
of albumin,
immunoglobulins-G and immunoglobulins-M, txl-antitrypsin , glycosaminoglycanes, C-reactive protein and electrolytes, and higher concentrations of immunoglobulins-A, antithrombin III, ceruloplasmin, 132-microglobulin , fibronectin, alkaline phosphatase and lactate dehydrogenase.
80 T
E 70 t3
65-
60 -2
-1
lg(tef) [s]
0
1
Fig. 5.19. Examples of tensiograms of various biological liquids obtained from a male patients, age 35, with reactive urinogenous chlamydial arthritis (Reiter disease ). Thick curve - serum, thin curve - synovial fluid, dotted curves correspond to average values for healthy males of corresponding age (serum) and for control group (synovial fluid).
Serum R-proteins are proteins of non-immunoglobulin nature which possess the catabolic activity with respect to peroxide oxidation reactions. They are present in the circulating blood both in the free form and as complexes formed with immunoglobulins-G, whose properties correspond to the naturally formed antibodies with various specificity (agglutinants or homoreactants.
Such complexes
reaches
up to 5% of the total
amount
of serum
immunoglobulins. For Reiter's disease the free fraction of R-proteins is reduced due to the
229 transition of these proteins into a form bound to immunoglobulins-G. This process is accompanied by a significant increase in the catalytic activity of R-proteins with respect to the peroxide oxidation reactions, a fact important for its destructive activity for arthritis (Salihbayeva et al. 1989). Note that the surface tension parameters of serum at t = 1 s and t --~ oo for Reiter's disease inversely correlates with the contents of serum immunoglobulins-G, and directly with the concentration of R-proteins. 5.7. Psoriasis
During the development of psoriatic arthropathy (PA), an improper microcirculation determined by rheological properties of blood in the articulatory synovia plays a certain role. For most of the patients with microcirculatory derangements, heavy hemorheological disbalances were found with an increase in plasma viscosity causing the hemorheological syndrome (Korotayeva et al. 1991, Wolf et al. 1981). The hemorheological syndrome develops via activation of fibrocytes located in the synovial membrane by such cytokines as interleukin1 that is released by the macrophagic-phagocytic system. Chronic inflammation of microvessels of the articulatory membrane is accompanied by hyperfibrinogenemia, degrading fibrinogen into the fragments D and E, which in turn stimulates the release of interleukin-1 by synovia macrophages. This haematological stress-syndrome is manifested by an increase in blood viscosity. In all cases of psoriatic arthropathy, the viscosity increases due to the increase in the concentration of high molecular proteins, mainly of fibrinogen. For psoriasis, the total concentrations of lipids, triglycerides, cholesterol, and phospholipids were increased, along with the variation in the contents of neutral fats and lipoproteins (Seishima et al. 1994, Imamura et al. 1990). The most significant changes of the lipid profile were observed for large-plaque type and pronounced exudative components of eruptions (Panasiuk 1988). For one-third of all patients a hyperuricemia develops due to the enhanced maceration of the epithelial cells and the output of nucleic acids. In addition, the purine exchange is deranged, which is related to the deficiencies of lipid metabolism (Sinyachenko & Barinov 1994). In spite of these significant changes in protein and lipid metabolisms, the dynamic surface tension parameters of serum for psoriatic arthropathy only slightly differ from those characteristic for healthy persons. There are, however, certain negative correlations between
230 and lipoprotein fractions of various densities, and also between ~ and the total contents of cholesterol in blood. 75
70
oo
65 I
~
I
~o ,,o
60
i
-2
-1
-
lg(tef) [s]
t 84
t
0
1
Fig. 5.20. Example for tensiograms of urine obtained from a male patient, age 37, with psoriasis, psoriatic arthropathy and nephropathy (male, age 37). Dotted curve correspond to average values for healthy males of corresponding age. It was rather unexpectedly found that significant variations of surface tension parameters in urine were found for patients with this disease (increase of t~2 and (r3, and increase of ~, see Fig. 5.20). It is quite possible that the genesis of surface tension variations is local (renal), in spite of ostensibly "intact" kidneys for psoriatic arthropathy found in the course of clinical, laboratory and instrumental studies. The alterations in kidneys during psoriatic arthropathy are manifested by segmental proliferation of mesangial cells, the increase of mesangial matrix volume, dystrophic changes in the epithelium of convoluted tubules, and interstitial oedema. For serious cases of this disease, the extent of damage suffered by glomerular and tubulostromal component increases, correlating with the character of articular syndrome (Shlopov & Shevchenko 1993). Most observations show the typical picture of mesangioproliferative glomerulonephritis. In some glomerules a symphysis of peripheral capillary loops with Bowman's capsule takes place, a focal proliferation of the epithelium of the external layer, with a possible subsequent sclerosis. T-helper-inductor lymphocytes prevail in the periglomerular cellular infiltration. The
231 tubulointerstitial component is characterised by the presence of protein masses in the tubular lumina, focal proliferation of the tubular epithelium, lymphoid infiltrates in the stroma with the presence of macrophages of haematogenic origin. The role of the antigen-antibody complexes in the development of psoriatic nephropathy deserves special attention, because the mechanisms responsible for glomerular filtration promote the retention and deposition of circulating immune complexes in kidneys. This effect is related to the existence of the receptors for the C3-component of the complement and F3-fragment of immunoglobulin-G at the epithelial and mesangial cells. Immune complexes are deposited in kidney glomerules due to the presence of antigens, similar to those in the glomerular basal membrane. All these factors lead to the angiopathy at the microcirculatory level, the increase in the contents of immunoglobulins (in particular, immunoglobulin-A), and to the activation of the complement system (Panasiuk et al. 1990, Imai et al. 1995, Yamamoto et al. 1994). 5.8. Gout
Among the dynamic surface tension parameters of serum sampled from patients with gout, only the ~ values decreased compared to healthy controls, despite the fact that this disease involves major irregularities in the composition of serum proteins (immunoglobulins, circulating immune complexes, ferments, hormones), lipids, non-albumin-type nitrous compounds, electrolytes and other surface active substances. Hyperuricemia is the most demonstrative evidence of gout. It was already mentioned that uric acid could affect surface tension parameters of a biological liquid. Similarly to other products of purine metabolism, uric acid correlates with the contents of sex hormones and gonadotrophic hormones in blood. For gout, the concentrations of these hormones undergo significant changes. In turn, the contents of proteins in blood depend essentially on androgens, estrogens and progestins. The metabolism of purines is closely related to the metabolism of fats. In 74% of gout cases a hyperlipoidemia was detected (types IIB and IV predominate), demonstrated by increased total concentrations of lipids, cholesterol, triglycerides and the low density lipoprotein fraction. The hyperlipoidemia is caused by an enhanced production of cholesterol-rich apolipoproteins and
232 triglyceride-rich very low density lipoproteins, and also by a decreased rate of degradation of very low density lipoproteins due to a suppressed activity of lipoprotein lipase, hepatic triglyceride lipase, letinin cholesterol transferase. Sytematic studies of various clinical forms of nephropathy complicating gout enabled us to differentiate between 3 types of this disease, which provide the most comprehensive description of the developments of the disease and its prognosis: urolithic, latent and proteinuric gout. The characteristic features of damages of the main kidney structures for various types of nephropathy are roughly the same, although some distinctions exist. The urolithic type is more often accompanied by damages of tubules, the proteinuritic type by damages of glomerules, while for the latent type damages of stroma dominate. Only in the urolithic type of nephropathy small kidney stones from calcium salts were found. Plasmocytic infiltration of kidney stroma occurs in urolithic and proteinuritic types of nephropathy. Plasmatic infiltration of the vessels occurs in the proteinuritic and latent types. The most significant extent of damages to kidney structures was found for patients suffering from the proteinuritic type of the disease. The lipid profile for the proteinuritic version of gouty nephropathy is characterised by hypercholesterolemia and hypertriglyceridemia, an increase of the cholesterol contents in the low density lipoprotein and very low density lipoprotein fraction, and a decrease of the amount of high density lipoproteins in serum. At the same time, increased concentrations of apolipoprotein-B, apolipoprotein-C and apolipoprotein-E, and lowered concentrations of apolipoprotein-A 1 and apolipoprotein-A 2 appeared. The development of chronic renal insufficiency leads to an increased production of low density lipoproteins and a slowdown of their degradation. It follows from our data that for gout (especially for female patients) the concentrations of calcium, either ionised, total or corrected with respect to albumin, in the peripheral blood is decreased, while its clearance is significantly increased. This effect is accompanied by an increase in the concentrations of parathyroid hormone, calcitriol and calcitonin. For the chronic form of gouty arthritis the levels of parathyroidin and calcitriolum are twice as high as those characteristic for the intermittent form. The most significant variations in parathyroid hormones were observed for patients with urolithiasis, cases where the most pronounced
233 disturbance of calcium homeostasis was observed. The indications of calcitonin in blood for the urolithic and latent types of nephropathy are the same, and do not differ significantly from those characteristic for healthy persons. On the contrary, for the proteinuretic version of kidney pathology the level of this hormone even increases significantly.
0 . 6
-
0.50.4E
0.3-
o
0.20.1 -
.~
0
m
::
-~ -0 1
iD~:~il I
o -0.2 k9 -0.3 -0.4 -0.5 TCa
ICa
PH
CTr
CT
Fig. 5.21. Correlations between the parameters of calcium and purine metabolisms for patients suffering from gout. Parameters of calcium metabolism are: TCa - total calcium; ICa - ionised calcium; PH - parathyroid hormone; CTr - calcitriol; CT- calcitonin. Parameters of purine metabolism are: hatched - uric acid, black - oxypurinol, white - xanthine oxydase, grey - adenosine desaminase
A direct correlation was found between the concentration of ionised calcium and the parameters of oxypurinolemia, and also a trend in an inverse correlation of total calcium in blood with oxypurinol, of calcitonin with oxypurinol, uric acid and adenosine desaminase (Fig. 5.21). High parathyroid hormone concentrations lead to a decreased tubular reabsorption of phosphates, resulting in a hypophosphatemia with subsequent compensatory mobilisation of calcium from the skeleton, accompanied by the development of hypercalciemia and hypercalciuria. It can be thus concluded that the qualitative and quantitative compositions of both surface active and surface inactive substances in blood for gout depend on the concentration of various hormones that are sometimes associated specifically with different forms of nephropathy. In this connection, a comparison study has been performed of dynamic surface tensiographic
234 parameters of serum obtained from patients with different versions of kidney lesion. For the proteinuric type, the decrease of k becomes yet more significant, while other surface tension parameters remain virtually constant. It should be noted that intermittent gouty arthritis is accompanied by the trend to decreased equilibrium surface tension of serum, while for the chronic version an increase was observed. The ~ values of serum tensiograms have opposite direction, and the values for the chronic form of the articular syndrome is two times lower than those characteristic for the intermittent form. It can be supposed therefore that the increase in the equilibrium surface tension and the decrease of L of blood for patients with gout can be regarded as evidence for the transformation of intermitting arthritis into the chronic form. Nephropathy is one of the most common visceral symptoms of gout. In fact, the disease is always associated not only with articular lesions, but also with renal lesions. Disturbances of renal functions in special examinations can be found even when no obvious clinical symptoms of nephropathy exist. Morphological changes in kidneys were detected by optical spectroscopy for all gout patients. The lesion of glomerules and stroma occurred in 100% of cases, tubular lesion in 88%, and lesions of vessels were found in 73% of cases. Most typical variations in glomerules are characterised by focal thickening of basal tubular membranes, an increase of mesangial matrix therein, and by focal (or, less common, diffuse) proliferation of mesangial cells, which are more or less pronounced. Glomerular sclerosis and hyalinosis can often be found. These changes, in 80% of all cases, resemble focal mesangioproliferative glomerulonephritis, while for the other 20% a similarity with mesangiocapillary glomerulonephritis was found. At
the
basal
membranes
of glomerular
capillaries
a
subendothelial
fixation
of
immunoglobulins-M and C3-component of the complement prevail. For this form of kidney lesions, a small increase of the mesangial matrix is typical. Weak proliferative reactions of mesangial cells can be explained by the minimum nephrotoxical ability of immunoglobulins-M as compared to other immunoglobulins. The glomerular depositions of immunoglobulins-G and complement are seldom detected, accompanied by a yet pronounced increase of the mesangial matrix and the creation of synechiae in the Bowman's capsule cavity. The occurrence of fibrosing mesangial cells for this type of gouty nephropathy is caused by the
235 stimulating influence of immunoglobulins-G on mesangiocytes, and by the hypoxia of tissue which takes place due to violations of the glomerular bloodstream. Electron microscopy studies show a proliferation and pronounced swelling of endothelium in some capillary loops, a proliferation and activation of mesangial cells with the tendency towards an interposition of their processes between the endothelium and basal membrane of the capillary loop. In many podocytes an expansion of cisterns of the granulated endoplasmic network is observed, accompanied by the loss of ribosomes. In uric space separated cellular organelles, fragments of the membrane and cellular cytoplasm can be found. In addition, podocytes contain a number of ribosomes and polysomes, and a hyperplasia of granulated endoplasmic network occurs, which is indicative of the increase in their metabolic activity (in particular that responsible for the synthesis of albumins). At some sections of the capillary loops the fusion of small podocytes, and the destruction of cells is seen.
7472 ,---, 7 0 -
{
68
~ 66 64 6260---2
~176
f
-1.5
-t
-
-1
t
-0.5
t-
--%-
f
0
0.5
1
--I
----q
1.5
2
lg(tef) IS] Fig. 5.22. Examples for urine tensiograms obtained from patients with gout and chronic arthritis, one with additional urolithic type of nephropathy (male, age 58, thick line), one with additional latent type of nephropathy (male, age 44, thin line); dotted curve correspond to average values for healthy males of corresponding age.
Dystrophy, atrophy and desquamation of the epithelium in tubules are determined, the expansion of lumen, hyalinosis and a thickening of the basal membrane, albuminous cylinders, leukocytes, small kidney stones from calcium salts and deposits of uric acid crystals are observed. The sclerosis of interstice is immanent to gouty nephropathy. The lesion of stroma
236 also demonstrates itself in the infiltration by lymphocytes, histiocytes, plasmocytes with giant cells. These effects are accompanied by great changes in vessels (sclerosis, proliferation of endothelium, lumen arctation, mioelastofibrosis, plasmatic steep, mucoid and fibrinoid swelling.) For gout, crl for urine increases. However, for the urolithic type strong incresing surface tension values of urine in all studied surface lifetime ranges result (Fig. 5.22). The latent type of nephropathy is accompanied by significant increases of the tensiographic parameter ~,. For the urolithic type no changes of ~, is obtained. For urolithiasis combined with gout, normal or2 values and slightly increased equilibrium surface tension were observed. 5.9. Osteoarthrosis Dynamic surface tension of urine sampled from patients with osteoarthrosis is quite similar to that characteristic for gout, while morphological changes in kidneys are absent. We believe that the increase in crl, cr2 and ~r3 values can be explained by the enhanced urinal excretion of glycosaminoglycanes, whose levels correlate with surface tensiometric parameters. It can be argued that the development of pathological processes in articulations for osteoarthrosis are characterised by disturbances in the metabolism of proteoglycans, which are the carbohydratecontaining polymers which play an important role in the construction of the connective tissue. The progressive dismetabolism of this tissue leads to a sharp decrease of the proteoglycans contents in the synovial media, while glycosaminoglycanes are the products of their degradation. High levels of glycosaminoglycanes in urine are related to the superactivity of [3-glucuronidase in blood (Astachova et al. 1987). In general, the destruction of the articular cartilage is determined essentially by the activation of hydrolytic ferments, which participate in the degradation of the main substances of the connective tissue: lysosome hydrolases and neutral proteinases, which include 13-glucuronidase (Gardner 1994). No dependence exists between clinical-roentgenological peculiarities of osteoarthrosis and glycosaminoglycaneuria, which can be explained by the heterogeneity of glycosaminoglycanes, represented by low-molecular forms, and also by non-dialysing high-molecular forms (Kosjagin et al. 1988). At further stages of the pathologic process, the dynamic surface tensions of urine increase. We can therefore
237 conclude that the surface tensiometry can be regarded as a better indicator of the progress of osteoarthrosis, than the contents of glycosaminoglycanes in urine. For patients suffering from osteoarthrosis, the synovial fluid contains extremely low amounts of immunoglobulins and 132-microglobulins, no fibronectin and c~2-macroglobulin, and low concentrations of lysosomic enzymes. At the same time, crystals of calcium pyrophosphate and hydroxyapatite are often present. We presume that these calcium salts contribute to the variation of surface tension parameters of synovial fluid during osteoarthrosis. This hypothesis is based upon the following findings: -
calcium ions in aqueous solutions of low molecular surfactants can increase the surface tension in the short surface lifetime range only;
-
low levels of albumin in synovial fluid only slightly affect the quantitative surface tension parameters;
-
calcium salts, which possess negative charges, promote the activation of factor XII (Hageman factor) in articulation cavity; this factor, in turn, activates kinins, low molecular mediators of inflammation, whose formation takes place via thrombin and plasmin, through the action of prekallikrein and high molecular kininogen;
-
the absorption of hydroxyapatite and calcium pyrophosphate by articulation cartilage chondrocytes leads to a significant release of neutral protease, collagenase (which is extremely active with respect to type I and II collagens) and adenosine triphosphate pyrophosphohydrolase; crystals of calcium salts lead to the increase of proteoglycans and mucin in the synovial fluid.
5.10. Effect of glucocorticoid therapy and plasmapheresis
The data concerning the effect of a glucocorticoid therapy on the state of surface tension for patients with glomerulonephrites are presented in chapter 4. It was mentioned that there is a trend towards a normalisation of dynamic tensiographic parameters during glucocorticoid therapy. Figure 5.23 shows variations of surface tension parameters of serum before and after administration of hormones to patients with rheumatoid arthritis, systemic lupus erythematosus and sclerodermia systematica. It can be concluded that glucocorticoid therapy increases surface tensions of serum, especially for sclerodermia systematica. For the cases of rheumatoid arthritis
238 and sclerodermia systematica, this is true mostly for (Y3, while for systemic lupus erythematosus the important parameter is el. For patients with rheumatoid arthritis the hormonal treatment leads to a normalisation of cl and or2, while for sclerodermia systematica the normalised parameters are g2 and cr3. For systemic lupus erythematosus (patients who do not suffer from lupus glomerulonephritis were also incorporated in the screened group) increased surface tensions of serum were observed. Although clinical-laboratory effects have been obtained, increased deviations of the parameters from normal values characteristic of healthy individuals occurs. The above is true also for the equilibrium surface tension of patients suffering from rheumatoid arthritis, and for g l values with respect to patients with sclerodermia systematica. The reactivity of the organism for rheumatoid arthritis is characterised by the contents of serum macroproteins, which promote the deterioration of the rheological properties of blood, and a more rapid decrease of the compensatory potential of vascular tension. The application of plasmapheresis leads to a decrease in the levels of immune complexes and immune active proteins. During the medication, an unambiguous correspondence was found between the parameters characteristic to macroproteinemia, and the concentrations of immunoglobulins-M and circulating immune complexes (but not with concentrations of immunoglobulins-G, fibrinogen and C-reactive protein). RA
10-]
SLE
SS
-10 -15 ~1
~2
~3
cl
~2
~3
~1
c2
~3
Fig. 5.23. Changes in surface tension parameters measured in serum obtained from patients with rheumatic diseases before (black columns) and after glucocorticoid therapy (white columns). Changes are given in % compared to corresponding healthy controls. RA - rheumatoid arthritis, SLE - systemic lupus erythematosus, SS - sclerodermiasystematica.
239 The application of plasmapheresis to patients suffering from rheumatoid arthritis leads to higher surface tensions of serum. This effect is caused by the excretion of some proteinic surfactants from the organism, while the contents of macroproteins is decreased along with an increase of the parameters indicative of relative albuminemia. The requirement to balance a protein deficiency is one of the problems, which arise in connection with a plasmapheretic treatment. In this regard, the main attention is paid to the development of specific methods aimed at the removal of those components from the plasma which are considered to play a key role in the pathogenesis of rheumatoid arthritis, while the plasma being returned back to the patient. During a selective plasmapheresis, fibronectin, complement components, cryoglobulins, circulating immune complexes and cryofibrinogen are removed. As there are no significant changes in the levels of albumins, the total protein contents at the end of a series of plasmapheretic procedures is similar to its initial value. The comparison of surface tensiometric parameters measured before and after plasmapheresis once again shows that pathologic proteins affect the surface tension of serum for rheumatoid arthritis. The elimination of pathological proteins from blood during plasmapheresis is clearly accompanied by the return of surface tensiometric parameters to values characteristic for healthy persons. Kidney lesions accompany many rheumatic diseases. Comparisons of the ratio of surface tensions for serum and urine at t = 0.01 s and t ~ oo have been performed for patients with such lesions. It was found that diseases of the connective tissue (systemic lupus erythematosus, sclerodermia systematica, rheumatoid arthritis) are characterised by significant increases of these parameters,
while in metabolic
and degenerative
articulation diseases
(gout,
osteoarthrosis) these values were decreased, see Fig. 5.24. Low equilibrium surface tension ratios were found also for non-specific aortal arthritis and seronegative arterites. In contrast, the serum to urine ratio for ~, significantly prevails for non-specific aortal arthritis, two or more times exceeding the corresponding values in other groups of patients, see Fig. 5.24. To summarise, we believe that the study of dynamic surface tension of biological liquids as applied to rheumatology is of significant practical interest, due to its capability of providing a differential diagnosis and monitoring of the efficiency of therapy. With regard to the above analysis of correlations between interface tensiographic parameters and the contents of surfactants it is now possible to indicate some surfactants which affect the surface tension state
240
o f serum, urine and synovial fluid. In turn, the d y n a m i c surface t e n s i o m e t r y o f biological liquids for rheumatic diseases is capable o f p r o v i d i n g rapid and rather accurate reflection o f the total c o m p o s i t i o n o f surfactants, including pathological proteins and other c o m p o u n d s formed and a c c u m u l a t e d during the d e v e l o p m e n t o f the disease. A
1.1
1.05 1 ~, 0.95 0.9 0.85 0.8 HC
HM
HF
*
R
SLE
HM
HF
*
R
SLE
SS
SS
HV NAA RA
BD
RD
PA
G
RD
PA
G
OA
OA
3 2.5 2
,
0.7
Most pronounced is the significant decrease of ~; during the treatment it approaches the characteristic value for the reference group of patients. The extent of ~. decrease can possibly be considered as one of the criteria of an efficient treatment, keeping in mind also the correlation between the value of X and the severity of the disease. As surface tensions depend on the composition of the liquor, we have analysed the correlations which exist between the concentrations of protein, glucose or chlorides, and dynamic surface tension parameters (cf. Table 7.7). It was found that for purulent and serous meningitis the correlations between similar values are often opposite to each other. For example, the extent of proteins level in cerebrospinal fluid for serous meningitis displays a pronounced positive correlation with ~ value ( r - +0.86), while for purulent meningitis this correlation is negative (r= -0.71). In addition, correlations of surface tensions with the cellular composition of liquor were analysed. Liquor cells do not influence the surface tension values directly; however, they affect the level of ferments and other synthesised substances, by which the surface tensions of liquor
316 can be influenced. It was found that the value of
(Yl
is closely related to the neutrophilosis of
cerebrospinal fluid for patients suffering from purulent meningitis (negative dependence), while the cy2 value for serous meningitis correlates with the lymphocytosis parameters (positive dependence). As the quantity of neutrophiles and lymphocytes in liquor is essentially characteristic for various types of meningitis, the application of surface tensiometry enables one to access implicitly the cellular composition of the liquor and therefore to perform a differential diagnostics of meningitis. The composition of cerebrospinal fluid has its peculiar features depending on the disease. For infection, increased concentrations of ceruioplasmin, fibrinogen, immune complexes, C-reactive protein, cholesterol, interleukin-6, lactate, acid phosphatase, isoferments - lactate dehydrogenase and kininase-II (hypertensin-I), glutamine, methionine, phenyl alanine, histidine, 7-aminobutyric acid, calcium were found, whereas for high density lipoproteids, C3and C4- components of complement the concentrations decreased (Dequette & Charest 1986, Dougherty & Jones 1986, Laurent & Schott 1986, Luca & Hategan 1986, Pitkanen et al. 1986 and Hashim 1995). The studies of liquor are believed to be important for the diagnostic of multiple sclerosis (gradual infection of the nervous system), as these can be used to estimate the intrathecal humoral immune response, which is the main constituent of the pathogenesis of the disease (Sch~idlich 1990). The specific form of leucine aminopeptidase exists in the cerebrospinal fluid of patients with multiple sclerosis, having physicochemical properties (molecular mass, optimum pH value, substrate specificity, electropheretical mobility) different from other isoforms of the ferment, also present in the liquor (Chochlov et al. 1987). The activity of [3-glucuronidase, neutral and acid proteinases in liquor was measured by Halonen et al. (1987). While changes in the concentration of lysosome hydrolases in cerebrospinal fluid are unspecific for multiple sclerosis, distinctive features of this disease are the increase in the concentration of neutral proteinase and a decrease in the concentration of acidic proteinase. The acute phase of multiple sclerosis is characterised by increased amounts of Oil-acidic glycoprotein (Tsukamoto et al. 1986) and circulating immune complexes, which comprise immunoglobulins-G, immunoglobulins-M, complement and [32-microglobulin (Procaccia et al. 1988). Both the level
317 of 132-microglobulin and the decrease in the index of C9-component of the complement show a direct dependence on the activity extent of the pathological process (Compston et al. 1986, Bjerrum et al. 1988). In 57 to 96% of all multiple sclerosis cases, the oligoclonal fractions of immunoglobulins, represented mainly by immunoglobulin-G with prevailing light n-type chains, were found in cerebrospinal liquor. For 46-78% of all cases, total hyper-7-globulinemia had developed (Ganes et al. 1986, Safar et al. 1986). With a decreased quantity of synthesised immunoglobulin-G, the oligoclonal spectrotype of immunoglobulin-G becomes more complex. Prolonged and progressive multiple sclerosis is accompanied by low amounts of immunoglobulins-G in the liquor, with a number of anomalous oligoclonal bands characterising the permittivity of the blood-brain barrier for albumins (Livrea et al. 1987). The data reported by Grucker et al. (1989) indicate an increase of the immunoglobulin-G index for 3% of healthy persons, 27% of patients suffering from central nervous system pathology, and 82% of patients with multiple sclerosis, with an increase in local
synthesis of
immunoglobulins-G found for 3%, 55% and 81% of all cases, respectively. The corresponding proportions for oligoclonal immunoglobulin-G are 0%, 79% and 17%, while the increase in the local production of immunoglobulin-M was found for 5%, 58% and 30% of all cases, respectively. With respect to the frequency of immune disorders, the presence of free light chains in cerebrospinal fluid for multiple sclerosis is second as compared to the presence of immunoglobulin-G oligoclonal bands, see (Bracco et al. 1987). This is possibly the reason why the correlation dependence of surface tension parameters for liquor on the level of immunoglobulin-G, immunoglobulin-A and C-reactive protein was found only for patients suffering from infectious pathologies. Amarenco et al. (1987) have studied the level of immunoglobulin-G and C3-component of the complement in cerebrospinal ~fluid for the Guillain-Barr6 syndrome (acute primary idiopathic polyradiculoneuritis). Local synthesis of immunoglobulin-G in the central nervous system for the stabilised paralysis T2 phase was higher than that in the progressive paralysis T~ phase and in the motion recovery phase T3. No correlation was found between immunoglobulin-G and the characteristics of albuminemia. The decreased amount of the complement C3-component was observed, especially in T2 phase. The higher the production of immunoglobulin-G in the cerebrospinal fluid was, the more severe were the clinical symptoms of the Guillain-Barr6
318 syndrome. No correlations were detected between the extent of transudation (total concentration of proteins and albumins in cerebrospinal liquor), and the severity of pathological processes. The studies performed by Papen & Warecka (1989) had shown that only for 10% of patients with Guillain-Barr6 polyradiculoneuritis, a synthesis of immunoglobulins in cerebrospinal fluid took place, and 67% of such patients exhibit altered blood-brain barrier functions. This course of the disease is characterised by significant (tens times) increases in C3a- and Csa-components of the complement, activated via the proteolytic splitting of C3- and Cs-components. It is worth noting that for vascular brain diseases, no changes of the complement system took place (Hartung et al. 1987). For vascular diseases of the brain, increased concentrations of alkaline phosphatase, lactate dehydrogenase, ATPase, ~/-glutamiltranspeptidase, creatine kinase, aspartate transaminase, adenylate kinase, serotonin, orosomucoid, alanine, potassium and etheric fraction of cholesterol is usual, with normal values of immunoglobulin-M and decreased concentrations of choline (c.f. Buttner et al. 1986, Nappi et al. 1986, Popova & Todorov 1986, Vrethem et al. 1987, Akimov et al. 1990, Manyam et al. 1990). It should be recalled that high inverse correlations between the amount of alkaline phosphatase and lactate dehydrogenase, and surface tension parameters of cerebrospinal fluid in the short time range was detected for vascular pathology only. Wester et al. (1987) had studied the level of monoamine metabolites and the activity of cholinesterase in the liquor of patients with brain blood circulation disorders. In 1/3 of all cases, an increase in the concentrations of 3-metoxy tyramine, homovanillin acid and 5-hydroxy indoleacetic acid was observed along with an increase in the level of spinocerebral fluid ferments (acetylcholinesterase and butyrylcholinesterase). The amount of serotonin and 3-metoxy-4-hydroxyphenyl glycol remains constant. A detailed analysis of surface tensiometric parameters for biological liquids with respect to brain and spinal turnouts is presented in Chapter 8; here it should be noted only that for oncologic diseases increases in the concentrations of 132-microglobulin, 13-globulins, fibropectin, astroprotein, and uric, lactic, pyruvic and fatty acids are observed with simultaneous hypopotassirachia and hypochlorinerachia. For patients suffering from neoplasm these amounts of 132-microglobulin and chlorine determine the surface tension parameters of liquor in the medium and long time ranges (inverse and direct correlations, respectively). The
319 concentration of creatine kinase in liquor becomes higher, and a correlation exists between the severity of the neurological symptoms and the amount of this ferment in cerebrospinal fluid (Matias-Guiu et al. 1986). The increase in the activity of ~,-enolase in liquor for brain tumours (especially for astrocytoma) indicates that a degradation of neurones occurs. It should be stressed that for slow infections, vascular diseases of the nervous system and craniocerebral traumas, the cerebrospinal fluid contains normal amounts of this ferment (Van den Doel et al. 1988). For 77% of all patients with nervous system neoplasm, an increase of the concentration of immunoglobulins in cerebrospinal fluid takes place (in 54% of cases - immunoglobulin-G, in 9% - immunoglobulin-M), while for 20% of patients, derangements in the synthesis of immunoglobulin-G were found (see Rao & Boker 1987). Increases in the index of immunoglobulin-M, oligoclonal immunoglobulin-G, 132-microglobulin and albumins in liquor were observed (Ernerudh et al. 1987). Antigenic heterogeneity of neoplasm, various localisation, the intensity of tumour growth lead to the formation of a great variety of antibodies and various rates of their influx into the cerebrospinal fluid (Hrazdira et al. 1987). Traumatic diseases of the central nervous system is the main source of primary damages of meninges, intermeningeal spaces and brain tissue. Irrespective of the trauma mechanism, a displacement of the brain takes place, which is inevitably accompanied by dynamic redistribution of liquor in the subarachnoidal space and ventricular system. Traumas of the central nervous system are often accompanied by epidural, subdural or subarachnoidal haemorrhages. Subarachnoidal haemorrhage is accompanied by the occurrence of blood in liquor; therefore the processes of blood coagulation, formation of clots and their lysis (at various stages of the disease) lead to the presence of a number of additional surfactants in the cerebrospinal fluid, which can affect surface tension parameters. It should be stressed that only the concentration of fibrinogen, which affects the coagulation ability of blood, correlates with cy2and ~3 parameters of liquor in cases of traumatic brain damage. The diagnostic significance of the determination of creatine kinase in cerebrospinal fluid for craniocerebral traumas is well known (Moshkin 1989). This ferment is contained in cells of brain tissue (astrocytes, dendrites, axons of neurone body), and during the first day after the
32O trauma its amount in the cerebrospinal liquor is increased. We have determined the activity of creatine kinase and its isoferments by adding 1 mg of the stabiliser ditiotreitol per 0.1 ml of biological liquid. The final concentration of magnesium in the reaction mixture was increased to 15 mmol/1, and the concentration of Trilon-B to 3 mmol/1. If the stabiliser was absent, then for brain damages reliable correlations do not exist between surface tensiometric parameters of cerebrospinal fluid and the concentration of creatine kinase. The addition of ditiotreitol results in a more frequent detection of isoferment BB with activity higher than 5 E/I, and in these cases a direct correlation exists between ferment concentrations and surface tensions of cerebrospinal fluid at t = 1 s and t ~ oo. Therefore, the application of dynamic surface tensiometry of cerebrospinal liquor may be considered as a reliable method for the estimation of the severity extent of traumatic brain damages. It should be noted that the concentration of fibrinogen and creatine kinase in cerebrospinal fluid is higher for open craniocerebral trauma, and correlates with the extent of brain injury. It is seen from Fig. 7.21 that the shape of tensiograms also depends on the character of the trauma. Moreover, direct correlations exist between the parameters of equilibrium surface tension for liquor and the amount of fibrinogen. 75 ~ 72 ' . . . . . . . . . . . .
o.o~
~
oo
~
~" 69 -~ t~ 66 ' 63 T I
~176
60 i . . . . . . . . . . . . . . . . . § . . . . . . . . . . . . . . -2
-1
+-. . . . . . . . . . . . . . . . . . . 4-. . . . . . . . . . . . . . .
q
0
2
Nt~f/[s]
1
Fig. 7.21. Examples for cerebrospinal fluid tensiograms obtained from patients with craniocerebral traumas and severe brain injuries. Thick line - open trauma (girl, age 5), thin line - closed trauma (boy, age 8); dotted line correspond to average values for control group.
321 7.6. Summary
In summary, dynamic surface tensiometry of serum and cerebrospinal fluid is useful for diagnostic and prognostic purposes and may have scoring potential to describe the severity of a given disease. We believe that further studies of surface phenomena in biological liquids taken from patients with neurological diseases should be extended into the following three areas. (a) The determination of unambiguous surface tensiometric parameters of biological liquids with respect to specific infection, vascular, spondilogenic, neoplasm and trauma related diseases should include patients sex and age; (b) The detection of surface active and surface inactive compounds which affect the state of surface tension of biological liquids, should include experimental in vitro studies employing the modelling of the composition of cerebrospinal liquor; (c) Estimation of the dynamic properties of surface tensiograms of biological liquids for neurological diseases should be given with respect to treatment and prognosis. 7.7. References
Akimov, G.A., Barsukov, C.F., Kurbatov, O.I., J. Nevrol. Psichiatr., 7(1990)3. Amarenco, P., Sauron, B., Schuller, E., J. Neurol. Sci., 80(1987)129. Barshtein, Yu.A., Yarosh, O.A., Persidsky, Yu.V., Vrach. Delo, 10(1989) 118. Bjerrum, O.W., Bach, F.W., Zeeberg, I., Acta Neurol. Scand., 78(1988)72. Bondarenko, T.I., Makletsova, M.G., Labor. Delo, 4(1992)12. Bracco, F., Gallo, P., Menna, R., J. Neurot., 234(1987)303. Buttner, T., Hornig, C.R., Busse, O., Dorndorf, W., J. Neurol., 233(1986)297. Chochlov, A.P., Baskaeva, T.S., Ckhrustaliova, N.A., Vopr. Med. Kchimii, 2(1987)58. Compston, D.A.S., Morgan, B.P., Oleesky, D., Neurology, 36(1986)1503. Dequette, P., Charest, L., Neurology, 36(1986)727.
322 Dougherty, J.M., Jones, J., Ann. Emerg. Med., 15(1986)317. Emerudh, J., Olsson, T., Berlin, G., von Schenck, H., Arch. Neurol., 44(1987)915. Ganes, T., Brautaset, N.J., Nyberg-Hansen, R., Vandvik, B., Acta Neurol. Scand., 73(1986)472. Gebesh, V.V., Muravskaya, L.V., Kononenko, V.V., Vrach. Delo, 10(1988)115 Grucker, M., Rumbach, L., Kiesmann, M., Sem. Hop., 65(1989)1253. Halonen, T., Kilpelainen, H., Pitkanen, A., Riekkinen, P.J., J. Neurol. Sci., 79(1987)267. Hartung, H.P., Schwenke, C., Bitter-Suermann, D., Toyka, K.V., Neurology, 37(1987)1006. Hashim, I.A., J. Ann. Clin. Biochemistry, 32(1995)289. Hrazdira, C.L., Hrazdirova, V., Polcakova, M., Ces. Neurol. Neurochir., 50(1987)238. Kapaki, E., Sogditsa, J., Papageorgiou, C., Acta Neurol. Scand., 79(1989)373. Laurent, B., Schott, B., Acta Neurol. Scand., 73(1986)477. Livrea, P., Simone, I.L., Trojano, M., Rev. Neurol., 57(1987)189. Luca, N., Hategan, D., Neurol. Psychiatr. Rev. Rom. Med., 24(1986)153. Manyam, B.V., Giacobini, E., Colliver, J.A., Ann. Neurol., 27(1990)683. Matias-Guiu, J., Martinez-Vazquez, J., Ruibal, A., Acta Neurol. Scand., 73(1986)461. Mendez, I., Hachinski, V., Wolfe, B., Neurology, 37(1987)507. Moshkin, A.V., Labor. Delo., 9(1989)48. Navarro, X., Segura, R., Acta Neurol. Scand., 78(1988)152. Nappi, G., Facchinetti, F., Bono, G., J. Neurol. Neurosurg. Psychiatry., 49(1986)17. Papen, R., Warecka, K., Psychiatr. Neurol. Med. Psychol., 41 (1989)334. Pitkanen, A.S., Halonen, T.O., Kilpelainen, H.O., Riekkinen, P.J., J. Nurol. Scand., 74(1986)45. Pokrovsky, V.I., Radsivill, V.I., Smysgova, A.V., Ther. Arch., 5(1989)130.
323 Popova, M., Todorov, V., Nevrol. Psikshiatr. Nevrokchir., 4(1986)6. Procaccia, S., Lanzanova, D., Caputo, D., Acta Neurol. Scand., 77(1988)373. Rao, M.L., Boker, D.-K., Europ. Neurol., 26(1987)241. Safar, J., Vymazal, J., Tichy, J., Ces. Neurol. Neurochir., 49(1986)382. Sch~idlich, H.J., Fortsch. Neurol. Psychiatr., 58(1990)247. Tsukamoto, T., Seki, H., Takase, S., J. Neurol. Sci., 75(1986)353. Tsvetanova, E.M., Liqvorologiya, Zdoroviya, Kiev, 1986. Van den Doel, E.M.H., Rijksen, G., Staal, G.E.J., Rev. Neurol., 144(1988)452. Vrethem, M., Ohman, S., von Schenck, H., Acta Neurol. Scand., 75(1987)328. Wester, P., Puu, G., Reiz, S., Acta Neurol. Scand., 76(1987)473. Yarosh, O.O., Vrach. Dielo, No 12(1991)58. Zaitsev, I.A., Arch. Clin. Exper. Med., 3(1994)215.
324
Chapter 8
Interfacial tensiometry in oncology Neoplasms are associated with compositional changes of blood. These variations change dynamic surface tension parameters tremendously. Therefore, dynamic interfacial tensiometry has potential concerning the diagnosis of certain types of tumours and monitoring of treatment.
8.1.
Pathogenesisof oncologicai disease
Patients with cancer of various organs have increased concentrations of 7-globulins, circulating immune complexes, [32-microglobulin, Otl-antitrypsin, haptoglobin, ferritin, orosomucoid, (z2glycoprotein, T-globulin, C-reactive protein and polyamines in blood (Guy etal. 1981, Mattison et al. 1981, Berdinskikh et al. 1987). An intensive synthesis of surface active fibronectin is performed by epithelial tumour cells, which leads to a hyperfibronectinemia (Titov & Sanfirova 1984). Amyloid P-component-glycoprotein is produced by hepatocytes. This glycoprotein is increased in serum obtained from patients with malignant tumours (Bannikova 1987). During oncological diseases, significant decreases in the level of vitamin-K-dependent glycoprotein C (molecular mass 62 kDa) in serum was observed (Ryabov et al. 1989). It was shown by Baskies et al. (1980) that direct correlations exist between the extensiveness of tumoural processes and the concentration of haptoglobin, orosomucoid and ot-antitrypsin in blood, while there are inverse correlations with the concentration of albumin, pre-albumin and ct2NS-glycoprotein. The ability of hepatoma to synthesise hepatic embryonic protein, ct-fetoprotein, was discovered thirty years ago. This fact strongly promoted the searches for new proteins, which arise during the development of neoplasms. The detection of oncofetal antigens can be regarded in some cases as an indication of tumoural development at its early stage, because the presence of these antigens depends on the degree of differentiation of tumour cells and the damage of intercellular links, and does not depend on the extend of the tumour. The characteristic feature of epithelial tumours is the increase of carbohydrate antigen 19.9 and carcinoembryonic antigens
(G[~I~ ~2-,
ct2H-, [~-, ~/1-, ,/2-fetoprotein, sulpho-glycoprotein) in
325 blood. The characteristic feature of tumours of the ovary is increased amounts of carbohydrate antigen 125, sialyl-SSEA, tissue polypeptide antigen, acid glycoprotein IAP and ferritin. The comedocarcinoma is accompanied by high levels of mucin-like glycoprotein and carbohydrate antigens 15.3 and 549. In serum obtained from patients with lung cancer, the concentrations of neurone-specific enolase and mucin-like glycoprotein are increased. Malignant tumours that arise from tissue that normally does not produce any hormones, often start hormone production. This secretion of hormones is often called ectopic secretion. It should be kept in mind in this regard that, under normal conditions the production of hormones happens not only in the endocrine glands, but also in the cells of so-called APUD (Amino Precursor Uptake and Decarboxylation) system. This (diffuse neuroendocrinal) APUD system consists of a complex of hormone-synthesising cells, specialised in the secretion of more than 35 various hormones and amines. It consists of neurosecretory cells of the brain, lungs, gastrointestinal tract, anterior lobe of the hypophysis, epiphysis, substantia medullaris of adrenal gland, C-cells of thyroid gland and D-cells of pancreatic gland. Therefore, the ectopic secretion is performed by cells which are neither endocrine cells, nor APUD system cells, and for which the production of hormones is not inherent. Table 8.1 summarises data concerning the ectopic production of hormones for malignant neoplasm of various localisation. Table 8.1. Ectopicallysecreted hormonesand localisationof tumours Hormone
Tumour localisation
Parathyroid hormone
Lung, mammary gland, kidney
Thyreotrophic hormone
Lung, mammary gland, chorionepithelioma
Gonadotrophic hormone
Lung, kidney
Somatotrophic hormone
Lung, stomach, uterus
Somatomammotrophin
Lung, intestine, mammary gland, uterus, thyroid
Somatoliberin
Lung, pancreas
Prolactin
Lung, kidney
Chorionic gonadotrophin
Lung, ovary, testicle, mammary gland, kidney, adrenal gland, liver, stomach, pancreas, intestine
326 The extracts of malignant neoplasm of lung, oesophagus and mammary gland contain much higher amounts of adrenocorticotrophic hormone and related peptides (13-1ipotropin, 13-endorphin, a-melano-stimulating hormone, enkephalins) compared to normal tissue. The synthesis of adrenocorticotrophic hormone and chorionic gonadotropin is common for all malignant tumours. Malignant neoplasm of the mammary gland is accompanied by a variations in the concentration of gonadotropic hormones in blood with an increased follicle-stimulating hormone level. The extent of such a hormonal imbalance depends on the stage of the disease and the size of the tumour. An inverse correlation was found between the level of the follicle-stimulating hormone, and the amount of prolactin, which possesses certain anti-gonadotropic properties (Agranat et al., 1991). Increased levels of calcitonin in blood was detected for the vast majority of patients suffering from lung cancer, pancreas carcinoma and struma maligna. The ectopic secretion of parathyroid hormone, together with osteoclast-stimulating factors and prostaglandin E2, results in a hypercalcemia for 10% of patients with malignant neoplasm. This fact was supported by tensiometric studies which have shown, that for lung cancer direct correlations exist between serum surface tensions in the short surface lifetime range, and the concentration of total and ionised calcium. However, no interrelation was detected between interfacial tensiographic parameters and the level of calcium-regulating hormones in blood. The somatotrophic hormone plays an important role in the regulation of carbohydrate and lipid metabolisms which is significantly disbalanced during tumourous processes (Dilman 1983). The increase in the amount of somatotrophic hormone in blood, and stimulation of lipolysis and ketogenesis during fasting indicates that somatotrophic hormone is contra-insular and acts as glucose-conserving agent. The hypersecretion of somatotrophic hormone in the organism of patients suffering from tumour increases the catabolic action of glucagon and corticosterone. The anabolic effect of somatotrophic hormone is hampered due to the decrease in the molar ratio insulin/somatotrophic hormone. The hypoglycaemic stress acts as a factor which initiates an increased secretion of the somatotrophic hormone. In addition, for patients suffering from oncological diseases somatotrophic hormone complements and enhances the biological effects produced by many other hormones (Shelepov et al. 1987).
327 Both the decrease in oxygen consumption by the tissue and the increase of lactate released into the blood directly correlates with the enhanced discharge of glucose (Bennegard et al. 1982, Edstrom et al. 1983). Thyroid hormones increase the rate of oxyhaemoglobin dissociation, which intensifies the transport of oxygen in tissue. The hypofunction of thyroid leads to a deterioration of this process, resulting in a tissue hypoxia. Usually a decreased glucose level in blood is observed for extended tumoural processes. Possible sources of hypoglycaemia are the ectopic production of somatostatin, somatomedin, proinsulin and insulin, the formation of insulinase inhibitors, retardation of glycogenesis in the liver, an increase in the glucose consumption by the tumour, intensification of glycolysis due to the suppression of lipolysis and the production of tryptophane (Dedov et al. 1988). Changes in the glucose concentration of a biological liquid can affect its surface tension, as it was demonstrated in Chapter 1. In fact, it was shown that increasing glucose levels in blood of patients with cancer is accompanied by increased surface tensions of serum in the short and medium time range. Interfacial tensiometric parameters for t ~ oo correlate negatively (however, less pronouncedly) with the glycemia level. The hormones secreted by any gland affect (directly or indirectly) other endocrine organs. Therefore, any imbalance of the endocrine equilibrium caused by tumours extends more or less to other endocrine secretion glands. Whatever preferential action is characteristic of the particular hormone (morphogenetic, metabolic or neurotropic), all the effects are based on the influence of hormones on the ferment system. Hormones can be involved in fermentative reactions as specific activators or inhibitors, which may result in a imbalance of the enzyme ~set)) of reacting cells. The main process in the invasion of malignant cells is the lysis of the extracellular matrix which acts as a barrier preventing the migration of invading cells. This lysis is performed by various ferments produced by the tissue. Proteinases are considered as the main enzymes (plasminogen activators, collagenase, cathepsins, proteoglycane-degrading ferments, elastase, gelatinase, etc.). A significant increase in the level of proteinases for tumours was observed (Geshelin et al. 1989, Varbanets 1990). The extracellular matrix is comprised of basal membranes and interstitial connective tissue. The main proteins of basal membranes are collagen type IV, laminine, and proteoglycans,
328 while the main proteins of the interstitial connective tissue are proteoglycans, fibronectin and elastin. In the cells of malignant phenotype, a high activity of plasminogen activators is observed, which transform plasminogen into plasmin. Plasmin splits laminine and fibronectin, and activates the latent forms of pro-collagenases. The activity of lysosome proteinases correlates with the rate of proteins metabolism in the tissue; therefore an increased activity in transformed cells can reflect the intensification of the intracellular metabolism (Sologub et al. 1992). Cathepsin B degrades collagen type I, laminine and proteoglycanes, and activates latent pro-collagenases of the connective tissue (De Bruin et al. 1988). The level of cathepsin H in brain tumours increases (Chernaya & Reva 1989). A similar increase of cathepsin D was observed in hepatoma cells by Maguchi et al. (1988). Cathepsin D intensively splits various proteins, including basal membrane proteoglycanes (Briozzo et al. 1988). In tumour cells and blood serum obtained from patients with malignant neoplasm, type IV collagenase is increased, which splits type IV collagen of the basal membranes (Kimura et al. 1990). The activity of this ferment correlates with the metastatic potential of tumours (Tryggvason et al. 1987). The components of the extracellular matrix are split by stromelysine - a ferment which is formed by connective tissue cells. Its primary structure is somewhat similar to that of collagenases, however, in contrast to these, stromelysine intensively degrades proteoglycanes and fibronectin, while it does not effect laminine, elastin and collagens. Elastase splits proteoglycanes, fibronectin, elastin and collagens of type III and IV. In the cells of carcinoma of the stomach, high molecular metalloproteinase (molecular mass ca. 1000 kDa) exists, which efficiently splits albumins, laminines, fibronectin, casein, haemoglobin and collagen type IV. The activity of this ferment remains unchanged in the presence of inhibitors of serine and cysteine proteinases (Tsuda et al. 1988). 8.2.
Serum tensiograms for different tumour localisations
The compositional changes of blood due to neoplasm depend on the localisation, size and histologic structure of the tumour. The total number of patients with malignant tumours studied was 165. Most changes in blood composition were observed for carcinoma of the stomach, lung and liver. At the same time, surface tension parameters of serum obtained from patients with carcinoma of the stomach (26 patients) show almost no differences in values of the
329
reference group (healthy persons). Lung malignant neoplasm (17 patients) was characterised by increased surface tensions in the short time range, while for liver malignant neoplasm (23 patients) equilibrium surface tension decreased, as shown in Fig. 8.1.
a) c~l, ~2, ~3 10-
I
_
J
0 ~ 9
-5
>
-10 -15 -20 St
Lu
Li
Ge
Mg
Br
Sc
Mg
Br
Sc
b)~, 100 80 60 ,-..,
40
~
2o
~9 .~. >
//
/
o
-20 -40 -60 -80 -100 St
Lu
Li
Ge
Fig. 8.1. Changes of surface tension parameters in serum obtained from patients with tumours of different location. Changes are given in % to corresponding healthy controls. St- stomach tumours, Lu- lung tumours, Li- liver tumours, Ge- genitals tumours, Mg- mammary gland tumours, Br- brain tumours, Sc - spinal cord tumours. The upper graph gives changes for crl - hatched, ~2 - black, cr3 - white. The lower graph gives changes for )~.
330 It should be mentioned that metastases in the liver following carcinoma of the stomach or lungs lead to a decrease in the equilibrium surface tension and an increase of ~ values of serum. In such cases, the interfacial tensiometric parameters approach those characteristic for primary hepatoma. These data let us conclude that decreases in equilibrium surface tensions of serum for patients with carcinoma of the stomach or lungs indicates metastatic spreading of the tissue into the liver, and represents evidence of the involvement of the liver in the formation of additional surfactants which can affect the dynamic surface tensions of biological liquids. For tumours of the female reproductive organs (42 patients) the lowest equilibrium surface tensions of blood serum was detected, and these changes do not depend on metastatic spreading into the liver. For neoplasm of genitals, the ~, values are increased up to values higher than those characteristic of patients suffering from primary carcinoma of the liver. It should be noted that the parameters of interfacial tensiograms virtually do not depend on the particular localisation of a tumour. No significant deviations from the normal amounts of the total protein in blood, its fractions, of cholesterol, triglycerides and lipoproteids of various density were detected; however, an increase in the level of some ectopically secreted hormones (in particular, chorionic gonadotropin and somatotrophic hormone), which can probably determine the serum surface tensions (either directly or indirectly via other surfactants) takes place. For malignant neoplasm of female reproductive organs, a significant increase of the [32-microglobulin in serum was observed. However, its concentration does not exceed a value characteristic for stomach or lung tumours, that is, for cases when either no change in the ~, values takes place, or these values become lower. In addition, no correlations exist between surface tension parameters and [32-microglobulinemia. For tumours of the mammary gland (21 patients) virtually no changes in averaged surface tension parameters of serum were detected. In some observations the dependence of equilibrium surface tension on the stage of pathological process was found, and correlations with some parameters of peroxide oxidation of lipids were detected which partly determine the composition of serum surfactant. This question will be discussed in more detail below. For brain tumours (26 patients) the values of gl and g2 decrease. The variations of these parameters for spinal cord neoplasm (10 patients) are still more pronounced, and the ~ value also becomes lower. For patients suffering from carcinoma of the stomach, liver and genitals,
331 inverse correlation exists between 9~ and equilibrium surface tension, while for spinal cord tumours this correlation is direct. For tumoural processes in the brain the behaviour of surface tensions of serum depends on the tumour location (Fig. 8.2). Serum
Liquor
1510-
o
0
2qu
.
-10 -15 2
3
4
5
6
1
2
3
4
5
6
Fig. 8.2. Changes surface tension parameters measured in serum and liquor obtained from patients with nervous system neoplsasms. Changes are given in % compared to corresponding controls. 1-accusticus neurinoma, 2- meningioma, 3- cerebellum turnout, 4- IV ventricle tumour, 5- posterior cranial fossa tumour, 6 - spinal cord tumour; hatched - 61, black -62, white - 63. For example, accusticus neurinoma is accompanied by low cl and c3 values of serum, while for turnouts of posterior cranial fossa these values are significantly increased. Equilibrium surface tensions for patients with meningioma becomes increased, while turnouts of cerebellum and ventricle IV lead to a decrease of this parameter. The localisation of pathological processes in the brain affects significantly the parameters of dynamic interfacial tensiograms not only for serum, but also for cerebrospinal liquid, as it is demonstrated in Figs. 8.3 to 8.5. Neoplasm of posterior cranial fossa are characterised by low 0.1 and 0.2 values with normal O"3 value of the liquor (cf. Fig. 8.2 and 8.3). Figure 8.4 shows the tensiogram of cerebrospinal liquid for patients suffering from ventricle IV turnouts. It is seen that the value of 0.3 is significantly lower, and the slope is higher, as compared with the reference curve.
332
a)
35 30 ,...,25 20
z15 ~1o 5
1
2
3
4
5
6
C
b) 300
I
200
~_~ 150 4 = r
!
"~ 100 t~
'
9
0 1 ~
--
-50
i
-100 J 1
2
3
4
5
6
Fig. 8.3. Z, values of biological liquids tensiograms obtained from patients with nervous system neoplasms.. 1 - accusticus neurinoma, 2 - meningioma, 3 - cerebellum tumour, 4 - IV ventricle tumour, 5 - posterior cranial fossa tumour, 6 - spinal cord tumour; C - control group. The upper graph gives ~, values in mN/m s ~/2, for serum (black) and liquor (white). The lower graph gives Z, deviation in % compared to corresponding controls.
W e have analysed the ratio of ~, values b e t w e e n serum and cerebrospinal liquid for brain tumours. W e found that this value remains normal only for tumours located in the posterior cranial fossa, see Fig. 8.6. For other types o f tumours, more or less p r o n o u n c e d decrease o f this p a r a m e t e r takes place.
333
7472,---, 70 68 66 64 6260 -1.5
-0.5
0.5 lg(tef) [s]
1.5
Fig. 8.4. Example for cerebrospinal liquid tensiogram obtained from girl, age 5 years with brain ventricle IV tumour; dotted curve correspond to average values for the control group
74 72 70 68
66 64 62 60
....
-
I
-1.5
-0.5
t
0.5 lg(tef) [s]
~176
1
1.5
Fig. 8.5. Example for cerebrospinal liquid tensiogram obtained from girl, age 3 years with cerebellum tumour; dotted curve correspond to average values for the control group
Table 8.2 summarises the variations in dynamic interracial tensiograms of biological liquids for patients with brain neoplasm.
334 Table 8.2. Differential diagnostic indicators of surface tension variation of biological liquids for different brain
neoplasm types Neoplasm Type Liquor
Serum (3"1
o2
o3
~,
a~
c:
or3
Accusticus neurinoma
-t-
Meningioma
+
+
Cerebellum tumour
+
+
iV ventricle tumour
~, -t+
+
Posteriorcranial fossa tumour
+
"+" - statistically significant increase of parameter compared to control; " - " - statistically significant decrease of parameter compared to normal
2.5
~9
2i 1.5-~ 1
0.5 l 0 --
// 1
2
3
4
5
6
C
Fig. 8.6. Ratio of serum to liquor tensiographic X values for nervous system tumours and control group; 1 accusticus neurinoma, 2 - meningioma, 3 - cerebellum tumour, 4 - IV ventricle tumour, 5 - posterior cranial fossa tumour, 6 - spinal cord tumour, C - control group.
It is seen that each kind of tumour has its specific features, which is important from a practical point of view. The differences in the dynamic surface tensions are caused by different compositions of surfactant in tumours and, in addition, can be determined by the age of
335 patients and the duration of the disease. For example, tumours of cerebellum, ventricle IV and posterior cranial fossa were characteristic primarily for children, whose liquor contains even for healthy children levels of ~,-globulins and [32-microglobulin lower than for adults, while the concentrations of amino acids are higher. In addition, the values of cy2, or3 and Z, inversely correlate with the duration of the disease, so that this factor should be considered in the analysis of interfacial tensiograms along with the patient's age.
8.3.
Correlation between surface tensions and biological liquid's composition
Dynamic surface tension parameters do not only correlate with turnout location but also with biological liquid composition. The presented data show that the ~2 value of liquor correlates directly with the total concentration of immunoglobulins, while ~1 depends inversely on the concentration of [32-microglobulin. The concentration of immunoglobulins-A in the cerebrospinal liquid taken from patients with benign neoplasm of posterior cranial fossa is usually higher than for malignant processes. For ca. 60% of adult patients with such a malignant turnout, hyper-immunoglobulin-A-emia was detected, while for children this number was three times lower. Similar data can be presented concerning the amount of immunoglobulins-G. The levels of immunoglobulins-M in liquor for benign and malignant tumours of posterior cranial fossa are roughly the same, and for children these levels are significantly higher than for adults (Rudenko et al. 1992). The protein composition of the cerebrospinal liquid for brain tumours is characterised by a significant increase in the amount of transferrin and high molecular ~2-macroglobulins (Vasilieva et al. 1995). No correlation was found between the dynamic surface tension parameters and the concentrations of transferrin and ~2-macroglobulin in cerebrospinal liquid. For patients with tumours of posterior cranial fossa, the concentration of transferrin in blood correlates positively with equilibrium surface tensions of serum. It was shown by Lampl et al. (1990) that the concentration of lactate dehydrogenase in cerebrospinal liquid increases even in the early stage of brain turnout processes. The dependence of interracial tensiometric parameters of biological liquids on the concentration of lactate dehydrogenase in liquor has been analysed and no reliable correlation was found between these parameters.
336 For
astrocytomas,
olygodendrogliomas,
glioblastomas
and
medulloblastomas
a
normoproteinrachia was observed, while for patients with accusticus neurinoma and corpus callosum neoplasm, a significantly increased protein concentration in cerebrospinal liquid was observed (Tsvetanova 1986). Inverse correlations were detected between the parameters crl and or3 of liquor, and the level of proteinemia. Therefore a more detailed analysis of dynamic interfacial tensiograms for various morphological versions of central nervous system tumours has been performed. Serum 20
Liquor
i
15 ~,
lO!
5 ~
oo!
'
U'
-5
-10 J -15 J Eb
Ar
As
Eb
Sert~
Ar
As
Liquor
250 200 1 r
150 t 100 ~
sol
~I-
-50
//
-100
Eb
Ar
As
Eb
Ar
As
Fig. 8.7. Changes of surface tension parameters measured in serum and liquor obtained from patients with various type of brain neoplasms. Changes are given in % compared to corresponding controls. Eb - ependymoblastoma, Ar- angioreticuloma, As - astrocytoma; The upper graph gives changes for ~ hatched, a2 - black, o3 - white. The lower graph gives changes for ~..
337 For astrocytomas, virtually no changes in surface tension parameters of serum were observed; for angioreticulomas 0.1 and 0.2 parameters for serum increased, while for ependymoblastomas these parameters decreased (cf. Fig. 8.7). In addition, ependymoblastomas are characterised by high equilibrium surface tensions and low ~ values for blood serum. The dynamic surface tension parameters of cerebrospinal liquid are also rather different: for ependymoblastomas a decrease of 0"2 takes place, for angioreticulomas a decrease of 0"2 and increase of 0"1 and 0"3 was observed, while for astrocytomas a significant increase of surface tensions in the short time range and a decrease of the equilibrium surface tension was detected. The 9~value for liquor for angioreticulomas was significantly lower, while for astrocytomas it was higher than the normal values. These data are of significant practical importance, enabling one to predict the morphological type of tumoural processes before a surgical treatment. Investigations were carried out to characterise the features of dynamic interracial tensiograms of biological liquids characteristic to patients with primary and metastatic neoplasm of the spinal cord. It was found that primary tumours of the spinal cord lead to a sharp increase of X for liquor, while for metastatic spreading into the spinal cord a decrease of ~ for serum takes place, as one can see in Fig. 8.8 and Fig. 8.9. These data are of certain practical importance for the differential diagnostics of pathological process in the spinal cord. Serum
Liquor
80 60 40 =
(D
20 0
-20 -40 -60 J 0.1
0.2
0.3
X
0.1
0.2
0.3
Fig. 8.8. Changes in surface tension parameters measured in serum and liquor obtained from patients with primary (black columns) and metastatic (white columns) various spinal cord neoplasms. Changes are given in % compared to corresponding controls.
338
75 70 65
.........
E
60 t~
55
45 -2
t
t
t
t
I
t
I
-1.5
-1
-0.5
0 lg(tef) [s]
0.5
1
1.5
Fig. 8.9. Example for serum tensiogram obtained from female, age 64 with tumour metastases into spinal cord; dotted curve correspond to average values for healthy females of corresponding age.
0.6 i
0.4 i
i
0.2
i
0 ~
-0.2
b ~)
-0.4
-0.6
i
-0.8 I
II
III
IV
V
VI
VII
VIII
IX
X
XI
XII
XIII
XIV
Fig. 8.10. Correlation coefficient between cerebrospinal liquid surface tension parameters and protein contents therein for patients with nervous system tumours. I - total protein, II - albumin, III - Oil-globulin fraction, IV - Ctx-globulin fraction, V - 13-globulin fraction, VI - ),-globulin fraction, VII - immunoglobulin-G, VIII - immunoglobulin-A, IX - immunoglobulin-M, X - ct2-macroglobulin, XI - 132-microglobulin, XII - fibrinogen, XII - transferrin, XIV - C-reactive protein; hatched - al, black -or2, white - cr3.
339 The variation in the composition of serum proteins is uncommon for neural system tumours. The disproteinemia in such cases, if any, is rather low. The correlation between interfacial tensiometric parameters and the concentration of serum proteins has been analysed and the results presented in Fig. 8.10. It can be concluded that concentration of proteins affects mainly the equilibrium surface tension values, and that a maximum correlation appears between equilibrium surface tension and the total concentrations of protein, albumins, 3,-globulins and [32-microglobulin. In the cells of human tumours (carcinoma of ovary, mammary gland, larynx) an albumin-like antigen (ALA, molecular mass ca. 68 kDa) was found (Bobrova et al. 1993). While the albumin contents in blood serum of patients suffering from oncological diseases, as measured using traditional methods, was usually found to be decreased (Lewis et al. 1991), the results obtained by immunoassay indicate an increase in the albumin concentration. The increase in the albuminemia level correlates with certain types of cells and the stage of the disease (Andersenn & Christensen 1991). Possibly for patients with malignant neoplasm, with respect to human albumin mouse antiserum detects an increased contents of some other antigens, which possesses crossed immune reactivity with albumins. With respect to some properties (thermoinstability, molecular mass, distribution in patients' serum) ALA is similar to cysteine proteinase (molecular mass 68 kDa), which acts as pro-coagulant, initiating blood coagulation (Gordon et al. 1990). The level of ferments in serum for patients with malignant tumours is much higher than for healthy persons, and corresponds directly to the stage of the disease. Qualitative and quantitative variations of serum albumin influence the surface tension parameters of blood exhibiting the most pronounced effects for patients suffering from hepatoma. The concentration of albumins inversely correlates with dynamic surface tensions in the short and medium time range, while there is a direct correlation with )~. During the development
of
the
nephrotic
syndrome
caused
by
paraneoplastic
membranous
glomerulonephritis accompanied by a significant decrease in the level of serum albumin, a decrease of cyl and
(5"2 values
was observed. This is illustrated by Fig. 8.11, showing the serum
tensiogram for female patients with carcinoma of the stomach, with a serum albumin concentration of 16.8 g/1.
340
72T "'~176176
.....
~ ~ 1 7 6 1 7 6 1 7 6 1. .7. 6. .
69 ~
............ ~ 1 7 6~ o
,
~
63
~
OOo
""
60 i -2
-1
0 lg(tef)[s]
t
t
1
2
Fig. 8.11. Example for serum tensiogramobtained from female patient, age 68 with carcinoma of the stomach, paraneoplastic membranous glomerulonephritis, nephrotic syndrome, chronic renal insufficiency 1st stage; dotted curve correspondto average values for healthy females of corresponding age. We believe that the variation in the surface tension of serum for malignant neoplasm is determined not only by the quantitative composition of albumins, but also by other factors: (a) variations in the qualitative composition of proteins caused by specific features of the metabolism related to the development of the nephrotic syndrome (cf. Chapter 4); and (b) the occurrence of hyperlipidemia (for patients suffering from carcinoma of the stomach, lungs and large intestine this is supported by a correlation between O'1 of blood serum and the amount of cholesterol). During oncological diseases serum albumin, which is highly surface active, undergoes pronounced conformational changes, caused by the increased load of ligands (Tolkacheva et al. 1991). In particular, extended binding of surface active poly-unsaturated fatty acids and products of peroxide oxidation of lipids by modified albumin occurs. For patients with malignant neoplasm, the total amount of lipids bound by albumin significantly exceeds that characteristic of healthy persons. This also depends on the location of the tumour and the extent to which the affected organ is involved in the metabolism of albumin ligands. The same fatty acids are present both in the albumin fraction of oncological patients and in albumin fraction of healthy persons; however, relative amounts of particular kinds of these compounds are essentially different, as one can see in Fig. 8.12.
341
250 200 1
il!ili
,.._150 = 100 ~9 50 0
--
-
-
.
.
.
.
.
-50 100 St
Pc
Li
EL
RP
Fig. 8.12. Changes of the amount of fatty acids bound to albumin in serum obtained from patients with malignant tumours of various localisations. Changes are given in % to values for healthy controls. St- stomach, Pc - pancreas, Li - liver carcinoma; EL - extraliver bile ducts, RP - retroperitoneal space; hatched-oleic acid, black-polyunsaturated fatty acids, white- c06-type polyunsaturated fatty acids, grey - 0~3-typepolyunsaturated fatty acids. Irrespectively of the location of the tumour, an increase is observed in the partial portion of 033 fatty acids which act as an important regulator of metabolic processes. The variations of the 0~6 series are less pronounced. However, for patients with neoplasm in the extrahepatic biliary tracts and retroperitoneal space, a significant reduction of the sum of basic fatty acids was observed. For patients suffering from oncological diseases, the increase in the concentration of oleic acid in the albumin fraction takes place (except for the cases when the tumour is localised in the extrahepatic biliary tracts). Both the oleic acid and poly-unsaturated fatty acids produce similar effects on some stages of the exchange process. As for oncological diseases the amount of poly-unsaturated fatty acids becomes significantly lower, it can be hypothesised that a substitution increase in the concentration of oleic acid happens in these cases. One of the important mechanisms for the support of homeostasis of the organism is the peroxide oxidation of lipids, which regulates the structure and function of biological membranes and their accessibility to various influences (Burlakova & Palmina 1982). The system of peroxide oxidation of lipids consists of activated metabolites of oxygen (superoxide anion-radicals, hydroxyl radicals, hydrogen peroxides, etc.), non-ferment and ferment antioxidants (superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase etc., cf.
342 Proter 1986). The formation and development of malignant neoplasm leads to changes in the intensity and character of peroxide oxidation of lipids in organs and tissue (Spector & Burns 1987, Kogarko et al. 1991, Korman et al. 1995, Gurevich et al. 1993, Gordon & Weizman 1993 and Casado et al. 1995), which reflects the mechanisms of the organism's reaction to emerging neoplasm (Potapov & Korman 1997; Korman et al. 1995). An integral parameter which enables one to estimate the intensity of peroxide oxidation of lipids is the total extent of unsaturation of fatty acidic components of lipids: the lower the number of double bonds, the more extensive is the oxidation by free radicals (Korman & Potapov 1994). The existence of tumours results in an attenuation of peroxide oxidation of lipids to an extent determined by the spreading of the process; for generalised tumoural processes this is accompanied by an increased proportion of unsaturated fats. Peroxide oxidation of lipids is a physiologic process required to support homeostasis and normal operations of the organism. The activation of the peroxide oxidation of lipids can be considered as a reaction towards the elimination of nosopoietic factors or as an adaptation to them, regulated by the system of anti-oxidant protection. In the initial period of the development of a tumour, a 'switching on' of the anti-tumoural protection mechanisms occurs, including the intensification of peroxide oxidation of lipids. However, at the same time an excessive oxidation by free radicals also becomes one of the pathogenic factors leading to a further development of pathological processes.
100
P
80 ~ i
6o ~ 40 -i ._o 20
o
0 4-
-20 f -40 " ,,
-60 J I
II
III
IV
Stage o f t u m o u r process Fig. 8.13. Levels of malonic dialdehyde and superoxide dismutase in serum obtained from patients with lung carcinoma (+% deviations from the parameters characteristic for healthy persons), black-malonic dialdehyde, white- superoxide dismutase
343 Figure 8.13 illustrates the level of malonic dialdehyde and superoxide dismutase in serum obtained from patients suffering from lung carcinoma at various stages. With the development of pathological processes, increased amounts of anti-oxidant ferments appear, which may be considered as the compensatory response to the peroxidation mechanisms. Simultaneously the surface tensions of serum are decreased (cf. Fig. 8.14).
MD
0.6
SD
0.4 -~ 9
0.2
o r..)
0
~
-0.2
o -0.4 -0.6 -0.8 I
II
III
IV
I
II
III
IV
Stage ofttmaour process
Fig. 8.14. Correlation coefficients between surface tension parameters and peroxide oxidation of lipids in serum obtained from patients with lung carcinoma of different stages. M D - m a l o n i c dialdehyde, SD - superoxide dismutase; hatched - a~, black -a2, white - a
The surface tension values in the short time range correlate inversely with the concentration of malonic dialdehyde, and directly with the concentration of superoxide dismutase. It appears that a2 for serum does not depend on the state of the peroxide oxidation system of lipids. -._
In kidneys malignant neoplasm, a strong intensification of the peroxide oxidation of lipids happen and result in an increased amount of malonic dialdehyde formed by both the ascorbatedependent and nicotinamide-adenine-dinucleotide-phosphate (NADP) -dependent mechanisms (Gorozhanskaya et al. 1995). One of the reasons for an enhanced peroxide oxidation of lipids is the low activity of protective ferments which destroy peroxides or prevents their formation (for example, the activity of superoxide dismutase is suppressed to a value two times lower than
344 that characteristic of normal conditions). It is generally believed that the formation of tumours is related to the decrease in the amount of superoxide dismutase in cells and even its complete disappearance. In addition, low catalase activity was found in tumours, caused by the suppression of the synthesis of ferments and the inactivation by formed hydroperoxides. One of the most important factors which regulate the peroxide oxidation of lipids is believed to be the amount of bioantioxidants in blood, which is significantly decreased during the progress of tumoural processes (Gorozhanskaya et al. 1989, Morozkina 1989). This is accompanied by either a decrease or an increase of a-tocopherol and retinol in tumours (Ostrowsky et al. 1989, Nikiforova et al. 1993). The amount of products of peroxide oxidation of lipids in serum albumin for patients suffering from oncological diseases is significantly higher than that for healthy people. However, when the tumoural process is localised in the liver or pancreas, increased concentrations of these products are accompanied by slight decreases in the amount of arachidonic acid, which is the main substrate of the peroxidation process. For carcinoma of the stomach, even an increase in the arachidonic acid amount was observed. In this case the activated formation of peroxides happens at the expense of other polyunsaturated fatty acids. The enhanced ability of serum albumin to bind peroxide oxidation products of lipids is considered to be a demonstration of the albumin antioxidant functions (Tolkacheva et al. 1995). In the albumins of patients with oncological diseases, essential decreases in the number of spiral structures and tyrosiles capable of perturbations was observed. The most significant variations in the structure of proteins were detected for liver tumours, which can be explained by a high contents of ligands. Therefore, for oncological diseases of various localisation, a pronounced structural change of serum albumins along with a decrease in the concentrations were observed. Large amounts of lipids, products of peroxide oxidation of lipids and polyunsaturated fatty acids of the
0) 6
family
in albumin is indicative of a disturbance of metabolic processes in the patient's organism, and also of large capabilities of the albumins to transport lipid-related ligands. Variations in the qualitative and quantitative composition of proteins and fats in serum affect its surface tension characteristics. The growth of tumours requires increased amounts of fats (Denisov et al. 1992). During the cultivation of cancer cells in the medium, depleted with respect to lipids, a significant decrease
345 in the concentration of cholesterol and phospholipids in cytomembranes was observed, while the ratio of saturated to unsaturated fatty acids amount had increased. The increase in the concentration of free fatty acids in blood serum and cell membranes is accompanied by the enhanced consumption of carnitine and the precursors of its synthesis, which is indicative of the increase in the utilisation of fats by the tumour. Unsaturated fatty acids possess a selective anti-tumoural activity, which can be blocked by antioxidants. The absorption of arachidonic and eicosapentaic acids by cancer cells decreases, while the absorption of linoleic acid remains unchanged. The excessive influx of fatty acids from cell membranes leads to growing concentrations in blood. High amounts of stearic and oleic acids in blood serum were observed for the carcinoma of the large intestine, while a higher concentration of arachidonic acid was detected for patients suffering from osteoblastoma, and higher levels of linoleic and a-linoleic acids was common for female patients suffering from breast cancer. Eicosanoids are metabolites of highly polyunsaturated fatty acids. These are prostaglandins, prostacyclins, thromboxanes, leucotrienes, various hydroxy- and hydroperoxy derivatives of fatty acids. The main sources for eicosanoids in the organism are believed to be linoleic, cx-linolenic and arachidonic (formed from linoleic) acids. The precursors of eicosanoids are contained in phospholipids and other complex lipids, which are surface active and determine the surface tensions of many biological liquids. It was shown in experiments that polyunsaturated fatty acids inhibit the synthesis of
prostaglandins E 2 and Fza in tumour cells, and these effects depend on the concentration of the acid and the particular acid species. The ranking of the acids is in the following order: with respect to prostaglandin inhibition: docosahexaenic > dihomo-y-linolenic > eicosapentaenic > a-linolenic > linoleic; with respect to a concentration decrease: dihomo-~,-linolenic > eicosapentaenic > docosahexaenic > c~-linolenic > linoleic. During the evolution of tumoural processes, significant disturbances take place in the eicosanoids metabolism and substantial changes were observed in the level of prostaglandins E 2, D 2, FI~, F2~ and thromboxane B 2 (Chiabrando etal. 1985, 1987). We believe that the
346 studies on the ferment activity in respect to the synthesis and catabolism of prostaglandins are very important. For example, the production of prostaglandin E2 in tumours is enhanced by a factor of 65 as compared to normal tissue, while for phospholipase A 2, which acts as catalyst for the release of arachidonic acid from phospholipids contained in the membranes, an eightfold increase was observed (Calo et al. 1984). It remains unclear whether during the development of a tumour the increase in the concentration of prostaglandins in blood reflects local or general disturbances of their metabolism (Kudriavtsev 1988). In many cases the removal of a tumour leads to a significant decrease in the level of prostaglandins E 2 and" E2~ for patients' serum. This effect, however, was observed only for certain localisations of pathological processes, and does not affect the amount of prostaglandin Fl,~ and thromboxane B 2 (Nigam et al. 1985). It was argued by Chaimoff et al. (1985) that high concentrations of prostaglandin E 2 in serum cannot be explained by its synthesis in tumour tissue. It was shown by Yoshino (1980) that for malignant tumours the level of low density lipoproteids in blood is lowered significantly, accompanied by higher amounts of fats and proteins. For female patients the type of lipidemia is related to the localisation of a tumour (uterus, mammary gland, intestine, stomach, cf. Kovalenko & Berstein 1993). Carcinoma of mammary gland is accompanied by increased concentrations of triacylglycerol, cholesterol and phospholipids, which exceed the corresponding values for patients with fibroadenoma and fibrous-cystic mastopathy (Araki et al. 1980). It should be noted that for the benign neoplasm of mammary gland, particular variations in the phospholipid contents were detected: increase of sphingomyelin with complete absence of lysophosphatidyl choline for patients with fibroadenoma, and an increase in the concentration of phosphatidyl ethanol amine together with decreased phosphatidyl choline contents for individuals with fibrous-cystic mastopathy. At the initial stage of a malignant growth lysophosphatidyl choline is also absent, while higher concentrations of serum sphingomyelin are found. A decrease in the concentration of phosphatidyl choline, and an increase in the amount of phosphatidyl ethanol amine is observed. The further development of a neoplastic process is characterised by a still more significant increase in the amount of sphingomyelin in serum (for patients with tumours < 4 cm in diameter) and phosphatidyl ethanol amine (for tumour diameters > 5 cm). For patients with
347 malignant tumours exceeding 6 cm in diameter, a suppressed anti-oxidant activity of blood lipids was observed. The application of radiotherapy results in a normalisation of the state of the anti-oxidant system and of the phospholipid levels: these characteristics become closer to those common for patients at early stages of tumoural processes (Kalnov & Palmina 1991). The irradiation leads to decreased concentrations of prostaglandins E 2 and F2~ in serum. These changes in the state of eicosanoids are accompanied by a reduction of peroxide oxidation of lipids and the activation of the anti-oxidant system (Shinjiro et al. 1989, Bilynskij et al. 1992). 8.4.
Influence of radiation therapy on dynamic surface tensions
For female patients with tumours of the reproductive organs, the application of a combined (remote and intracavitary) radiation therapy results in a decrease of the concentrations of malonic dialdehyde and dien conjugates in serum, while the amount of (z-tocopherol becomes higher. Such variations in the peroxide oxidation system of lipids correlate with the equilibrium surface tension. It is interesting that these variations are accompanied by a decrease in the interfacial tensiographic parameters of urine at t-~oo, see Fig. 8.15. At the same time, a pronounced decrease in X values of serum and urine results (Fig. 8.16). Therefore, an inverse correlation exists between equilibrium surface tension and the ~, values of serum, while for urine tensiograms these parameters are inversely related to each other. Serum tensiograms for patients suffering from uterus body, or neck carcinoma, which have been treated by remote radiation therapy are presented in Figs. 8.17 and 8.18. It should be noted that for healthy females no correlation exists between the values of ~3 and of different biological liquids, cf. Chapter 3. In this regard, the following suppositions can be made: 1) during radiotherapy for patients with reproductive organs malignant neoplasm, a decrease of surfactants (including the pathological ones) in blood happens, which leads to increased equilibrium surface tension values; 2) enhanced urinal excretion of some portion of blood surfactants leads to a decrease in the equilibrium surface tensions.
348
Serum
62 -t !
Urine
6o I
58 ~ ~" 56~ ~ 54 ~ 525048 ,~-
,~
~
,t
~
II
- - T
III
.
.
.
.
H
T - - ~
- - T - - ~
I
l
II
Serum
-
-
III
1
- -
H
Urine
. . . . . .
-2-4 -
9
-8-
"~ -10 -12 t 14 -16 I
II
III
I
II
III
Fig. 8.15. Influence of radiation therapy on equilibrium surface tension in serum and urine obtained from patients with genital tumours and healthy controls. Upper graph gives the equilibrium surface tension for patients and healthy controls in mN/m. Lower graph gives changes in % compared to healthy controls. I - before the treatment, II- after remote radiation therapy (22-26 Gy) with intracavitary radiotherapy (20 Gy), III- after remote radiation therapy (46-48 Gy) with intracavitary radiotherapy (40-50 Gy), H- healthy females
349
Senun
Urine
1, t 16
14 12lO,
