ELECTROPHORESIS

From Big Medical Encyclopedia

ELECTROPHORESIS — the directed movement electrically charged particles of a dispersed phase in a dispersion medium (or ions in electroconductive solution) under the influence of external electric field. The method of an electrophoresis is widely used in biology and medicine for allocation and the analysis of individual proteins (see), nucleic acids (see) and other biopolymers, viruses, supermolecular cellular structures, and also whole cells. In immunology one of the most used methods of a research is immunoelectrophoresis (see) — electrophoretic unmixing of antigens or antibodies in gel about the subsequent their precipitation. By a microelectrophoresis (see. Mikroionoforez ) it is possible to enter into a cell or to bring any substances capable to dissociate on to it ions (see). The microionophoresis is one of the main modern methods in neurophysiological, neuropharmacological, neurochemical researches. Great diagnostic value have electrophoretic division enzymes (see) on coenzymes (see) both their quantitative and quality standard. Administration of medicinal substances in an organism by E. it is widely applied in physical therapy (see).

AA. along with electroosmosis (see) it was opened in 1807 by professor of the Moscow university Reyss. Electrokinetic phenomena (see) to which carry E., are caused by existence on the interphase boundary of a double electric layer and ability of a diffusion part of this layer to be displaced it is rather adsorptive its connected (motionless) part. Electric potential of the surface dividing mobile and motionless parts of a double electric layer carries the name electrokinetic or ζ (zeta) - potential. The particles of a dispersed phase which are in buffer solution (see. Buffered solutions ), a certain boundary electric charge, size and a sign bear to-rogo depend on the size pH of the environment (see. Hydrogen ion exponent ). If through the buffer solution concluded in a vessel with electric insulating walls, napr in a glass tube to pass electric current, then emergence of a certain gradient of tension will be result of it (see. Gradient ), or electric field. Under the influence of this field of a particle of a dispersed phase according to a sign of a boundary space charge move in the direction of the cathode, i.e. there is a cataphoresis, or the anode — anaphoresis. Depending on the size of a charge and the sizes of a particle in electric field get different speeds. The mix of diverse particles brought in a narrow zone in these conditions is divided into the zones formed by the particles moving with an identical speed, i.e. having identical electrophoretic mobility.

Electrophoretic mobility of the particles having spherical shape (V) is expressed by Smolukhovsky's formula: V = (ζD)/(4πη), where ζ — the electrokinetic potential of the double electric layer surrounding a particle, D — dielectric permeability and η — viscosity of the environment. In that case when electrophoretic unmixing of particles (or molecules) is made in buffer solutions with not too low (e.g., apprx. 0,1) values of ionic strength of solution (a half-sum of works of concentration of all ions which are in solution on a square of size of their charge), particles are grouped in fractions only in size of a charge without the sizes or a pier. scales (masses) if it is about molecules.

Fig. 1. Electrophoretic division of serum proteins of the healthy person in the device Tizelius (optical recording). The arrow specified the direction of the movement of fractions in sweat of direct electric current in the borate buffer, pH 6,8. Separate peaks on an elektroforegramma (1 — 5) with reply γ-, β-, α2-, α1-to globulins and albumine of blood serum. Using the schedule, it is possible to calculate abundance of each fraction, having accepted the area limited to the basic line and a curve for 100%; «—» and «+» — the cathode and the anode.

Use E. in biology and medicine 20 century when A. Tizelius developed a method E began in the 30th. in free liquid also designed the device for electrophoretic division and the analysis of mix of proteins by a so-called method mobile, or free, borders. In a medico-biol. researches apply a set of options of two main modifications of an electrophoretic method — an electrophoresis in free liquid (a svobodnoprotochny electrophoresis) and a zone electrophoresis (a zone electrophoresis, or AA. on inert carriers). The first developed E. in free liquid (a method of mobile borders, an electrophoresis across Tizelius), to-ry allowed to measure electrophoretic mobility of the examinee of substance on movement of mobile border between the pure buffer solution and buffer solution containing the studied substance. In Tizelius's device the optical technique of registration of position of such border by determination of index of refraction of the environment is used (see. Nefelometriya , Refractometry ), and in nek-ry cases — a direct mikroskopirovaniye. During the unmixing of substances with various isoelectric points (see. Isoelectric point ) optical devices register several moving peaks (fig. 1). Main shortcoming E. in free liquid its thermal agitation which is preventing sharp separation of fractions and washing away borders of zones is. This shortcoming is partially overcome by creation of gradients of density of buffer solutions (e.g., by means of sucrose). At fractionation of low-molecular substances to avoid excessive washing out of zones, apply high-voltage E., sometimes in combination with chromatography (see) — a so-called method of «fingerprints».

Fig. 2. The scheme of the elementary electrophoresis apparatus in gel: and — prior to fractionation; — after the end of fractionation; «—» and «+» — the cathode and the anode; 1 — the working channel (glass tube); 2 — electrode tanks (through the buffered solution which is in them and the working channel the electric chain between electrodes becomes isolated); 3 — the place of entering into gel of the mix which is subject to electrophoretic division; cross strips within a brace — the zones formed as a result of an electrophoresis.

Zone E. differs from E. in free liquid mainly use of neutral supporting medium (inert carriers) for a liquid phase (buffer solution) that minimizes effect of thermal agitation and allows to allocate if necessary that site of the carrier, to-ry contains individual substance. As inert carriers in zone E. use special hromatografichesky paper, strips of cellulose acetate, thin coats of silica gel, powder of cellulose or gels of sefadeks (see. Dextran ). Zone E. on inert polymers carriers allows to fraction substances not only in size of a charge, but also on a pier. to weight. A specific place among such carriers is held by gels of polyacrylamide (PAAG) and agarose. Advantage of polyacrylamide gels consists in a possibility of change of diameter of their time at change of concentration of polymer, and also in lack of the phenomena of adsorption and electroosmosis at an electrophoresis.

At electrophoretic division of heterogeneous mix in polyacrylamide gel a column of small section (apprx. 1 cm 2 ) fill with the buffer solution containing the dissolved monomer (acrylamide; CH 2 — CH — CONH 2 , a small amount of substance stapling machine (encore - N-metilenmetakrilamida — HC(CH 2 ) — CONH — CH 2 - NHCO-(CH 2 ) CH) and substance - the polymerization initiator. Through a nek-swarm time at the room temperature in a column is formed homogeneous gel (fig. 2). If by means of E. in free liquid across Tieelius in blood serum find 5 protein fractions (see fig. 1), at electrophoretic division of blood serum in polyacrylamide gel total them not less than 25 (fig. 3).

Resolving power E. in polyacrylamide gel considerably raises during the use as the carrier of system of gels (usually two — «worker» finely porous and directly over it «creating» krupnoporisty). Except degree of porosity, these gels sharply differ in size pH and molarity of buffered solutions in which they will be polymerized. Taoka E. call step, or diskelektroforezy (English (discontinuous - discontinuous).

Option E. in polyacrylamide gel E is. mixes of biopolymers after pretreatment by the denaturant agent for the purpose of change of a configuration of molecules. Proteins in this case process ionic detergent (see) — dodetsilsulfaty sodium, destroying disulfide bridges in their molecules and forming with them negatively charged micelles, a charge to-rykh the pier is proportional. to the weight of protein; nucleinic to - you subject E. in the presence of alkali, urea, formamide or other agents destroying hydrogen bindings in polynucleotide chains nucleinic to - t. Under these conditions electrophoretic mobility of biomolecules begins to correlate strictly about their pier. it is powerful.

Fig. 3. The diagrammatic representation of an arrangement of serum proteins at paper electrophoresis or cellulose acetate (I) and in polyacrylamide gel (II): And — albumine; γ-, β-, α2-, α1 — Fractions of the corresponding globulins; pre-A — prealbumin; α1-ГП — α1-гликоиротеид acid (α1-серомукоид); AT — α1-antitrypsin; C - ceruloplasmin; TF - transferrin; - Hemopexin; PFL - plasminogen (plasminogen); IgA + IgG - A and G immunoglobulins; GP1 - β2-glycoprotein I; f - fibrinogen (a factor of the I coagulant system of blood); o2M - α2-macroglobulin; β-LP +IgM — β-lipoproteids ((((((((((lipoproteids of low density) + immunoglobulins M; GPT — gaptoglobin.

For overseeing by the course E. in gel add low-molecular dye, chemically inert concerning the divided substances to the studied mix (see. Dyes ), molecules to-rogo bear electric charge of the same sign, as a molecule of the divided substances, but have electrophoretic mobility, edges are slightly higher than mobility of the protein fraction which is moving ahead the first. Such dye call leading. Most often in alkaline and neutral buffer solutions use bromphenol blue, in acid medium — imperial green or pyronin. When the painted zone reaches the end of gel, AA. stop, after fixing for a certain time immerse gel in solution of specific dye then excess of dye wash (fig. 4) For identification on an elektroforegramma of proteins-enzymes sometimes use their catalytic activity concerning chromogenic substrates. Detection of electrophoretic zones on their radioactivity is widely applied (see. radiography ).

Many researchers as inert carriers prefer gels in the form of thin plates. AA. in a gel plate does more reliable comparison of separate drugs, allows to carry out two-dimensional division, etc. For the analysis of amino acids, peptides and sugars (in the form of their borate complexes) use high-voltage E. on paper, in a thin coat of silica gel, cellulose acetate and other dyes.

Division of complex mixture of proteins not always manages to be carried out even during the use of the listed above receptions E. Therefore in difficult cases apply a so-called two-dimensional electrophoresis when after the first electrophoretic fractionation of mix of proteins each strip is used as initial drug for an electrophoresis in the perpendicular direction in relation to the direction of the first division. As a result on the second plate there is a large number of the zones corresponding to individual proteins (sometimes their number reaches 2 thousand).

There are methods combining, e.g., an electrophoresis and chromatography (see); sometimes unmixing of proteins is carried out in the perpendicular directions, or in one direction of a squirrel divide E. in polyacrylamide gel with dodetsilsulfaty sodium, and in perpendicular to it — with the help isoelectric focusing (see). The last method allows to reveal on one gel plate to 7 thousand individual proteins. Options E. also an electrophoresis in a gradient of pH values and an electrophoresis in a gradient of porosity of gel, the immunoelectrophoresis, the affine electrophoresis combining advantages E are. and affine chromatography, etc.

By means of E. proteins define their primary structure, a pier. weight, pathogenicity and existence of multiple forms. For an electrophoresis of cells use svobodnoprotochny E. in its analytical and preparative options. So fraction bacterial cells, viruses, and also lysosomes, mitochondrions, Glldzhi's complexes and other cellular organellas. Molecules nucleinic to - t differ from molecules of proteins in a strong negative charge. Fractionation of their mixes carry out a pier due to distinctions. weight of native high-molecular DNA and RNA. For electrophoretic fractionation of their low-molecular fragments use krupnoporisty gels of agarose or gels of polyacrylamide with concentration from 5 to 20%, and also their mixes. The analysis of fragments nucleinic to - the t received during the splitting of molecules DNA by nucleases and chemical agents gives the chance to define primary structure of these biopolymers, i.e. to a strukter of genes (see. Gene ).

Fig. 5. The isofermental ranges of a lactate dehydrogenase (LDG) of blood serum received by method of an electrophoresis: and - the isofermental range of LDG are normal; — at an acute myocardial infarction; in — at a viral hepatitis; «—» and «+» — the cathode and the anode.

Method E. allowed to find normal hereditary polymorphism of proteins of the person. Tens of options of haemo globins became known (see. Gemoglobin ), glyukozo-6-phosphate-dehydrogenase and other proteins. Data on multiple forms of enzymes were obtained (see. Isoenzymes ), consistently expressed during ontogenesis and genetically independent. As a result of a blood analysis, urine, cerebrospinal liquid E. changes of a normal expression of the genes coding synthesis of certain proteins at various morbid conditions (fig. 5) were revealed.

By means of the electrophoretic analysis enzymes (see) diagnosis, including prenatal, some inborn diseases is possible.

At molecular pathology there is a change of density of a relative charge on a surface of cells therefore by method E. it is possible to reveal and divide subpopulations of B-and T lymphocytes, e.g.

Researches on receiving especially pure drugs (e.g., interferon) are begun with method E. in the conditions of zero gravity at space flights.

Medicinal electrophoresis

Medicinal electrophoresis (ustar. an ionophoresis, an iontophoresis, an ionotherapy, a galvanoionitherapy, an ionogalvanization) — the method of electrotreatment consisting in the combined impact on an organism of a direct current and the medicinal substances entered with its help. In medical practice medicinal E. it was entered since 1802 when Rossi for the first time applied to impact on an organism of the patient medicinal substances in combination with a direct current (see. Galvanization ). Long time for medicinal E. used only direct continuous current (galvanic). In a crust. time is widely applied by diadynamic currents (see. Diadinamoelektroforez ), the sinusoidal currents modulated (amplipulsforez) and fluctuating (flyuktuoforez) in the straightened mode.

Basic basis medicinal E. the theory of electrolytic dissociation is (see. Dissociation in chemistry , Electrolysis ). The medicinal substances capable to dissociate in solution on positive (cations) and negative (anions) ions, directionally move to the field of direct electric current and can come to an organism, breaking a skin barrier (see. Skin ). At the same time from electrode laying only those ions are entered, to-rye have the sign of the same name with an electrode.

At E. the main ways of penetration of medicinal substances to an organism through skin are output channels stalemate and, to a lesser extent, sebaceous glands. A part of medicinal substance gets into an organism through intercellular spaces and a part — through cells (especially at electrophoretic administration of medicinal substances through a mucous membrane).

At E. medicinal substances get on small depth: right after the procedure they are found generally in epidermis and a derma, in a small amount — in hypodermic cellulose. From here entered in the way E. medicinal substances arrive in lympho-and a blood stream and are carried on all organism though preferential they collect in fabrics and bodies of the area of influence.

AA. medicinal substances through skin and mucous membranes quantitatively do not submit to laws of electrolysis since living tissues have electrocapillary activity (see. Elektroosmos ) and barrier properties (see. Barrier functions ). At E. from only 1 to 10% of the substance which is in solution are entered into an organism (on laying). On quantity entered in the way E. substances significantly influence physical. - chemical properties of pharmaceuticals and property of their solutions (extent of dissociation of substance, the sizes, size and a sign of a charge of an ion, an opportunity and extent of its hydration, the used solvent, concentration, etc.), conditions of holding a physiotherapeutic procedure (density of current, duration of influence. age of the patient, etc.), functional condition of an organism in general and skin in particular.

The medicinal substance entered by method E. can affect an organism in the reflex way (a so-called ponny reflex on Shcherbaka), a humoral way and. besides, to have local action. It depends on type and amount of medicinal substance, a technique and conditions of holding a procedure, parameters of a physical factor, etc.

The electric current used for E., causes various physical and chemical, metabolic and cellular and fabric reactions in an organism (see. Galvanization , Diathermy , Diatermoelektroforez ), against the background of to-rykh action entered by means of E. medicinal substances gains a number of features and advantages in comparison with usual ways of pharmacotherapy (see). The greatest practical value at medicinal E. have the following factors:

  1. longer effect of medicine and slower removal it from an organism thanks to, first of all, education in skin of depot of ions, about patching pharmakol. activity;
  2. a possibility of creation of high local concentration of medicinal substance without saturation by it of blood and other environments of an organism;
  3. smaller probability of emergence of side reactions;
  4. administration of medicinal substance in most pharmacological to an active form — in the form of ions;
  5. painlessness of administration of medicines and lack of the deformation of fabrics arising at other ways of pharmacotherapy because of administration of solvent.

Thanks to a promoting effect of electric current the clear specific and expressed therapeutic action entered in the way E. medicinal substances it is shown at such concentration, to-rye at usual ways of pharmacotherapy would be ineffective or inefficient.

Appointment medicinal E. is defined, on the one hand, by favorable medical effect of direct continuous current or other types of electric current (see. Impulse currents ), and on the other hand — indications to use of the corresponding pharmaceuticals.

Medicinal E. it is impossible to apply when there are objective contraindications to use of electrotreatment and the corresponding pharmaceuticals, and also at their individual intolerance.

Equipment medicinal E. comes down to an arrangement on the way of current (between a body of the person and electrodes) solution of medicinal substance. Depending on a way of putting medicinal substance and leading of current distinguish several options medicinal E. Naiboley electrophoretic administration of medicinal substances from solutions is widespread, to-rymi special laying between a body of the patient and an electrode is moistened. Technology of performance medicinal E. in this modification differs from the equipment a little galvanization (see). The only difference is that electrode laying is moistened not with mains water as at galvanization, and solution of medicinal substance. This solution by means of the burette or other portioning device is quantitatively applied on hydrophilic laying or, more often, on the special medicinal laying located at the procedure between skin and protective laying. Medicinal laying is prepared from 1 — 2 layers of filter paper or 2 — 4 layers of a gauze. In a form and the area they shall correspond to protective laying. Moisten with solution of medicinal substance usually one laying, however the medicinal substances dissociating on ions with opposite charges laying can be applied on both (the cathode and anode).

Solution of medicinal substance is applied on laying of an electrode (positively charged — the anode or negatively charged — the cathode), of the same name with the ion which is subject to electrophoretic introduction. At the choice of polarity it is necessary to consider the following: ions of all metals, mestnoanesteziruyushchy means, the majority of alkaloids, antibiotics and sulfanamide drugs have positive charge therefore at an electrophoresis they shall be entered from the anode, and ions of all metalloids and acid radicals get a negative charge in solutions and, therefore, shall be entered into an organism from the cathode electrode. The boundary space charge of amphoteric connections (a squirrel, amino acid, etc.) depends on their ionic structure and the size pH of the environment (see. Hydrogen ion exponent ): at low pH values the charge becomes more positive, at high — more negative.

At so-called vannochkovy E. immerse the naked part of a body of the patient which is subject to influence in the tray (glass, faience, plastic) with the built-in electrodes filled with solution of medicinal substance.

Polostna medicinal E. is that before introduction of the electrode connected to the corresponding pole of the device for medicinal E., in a gastric cavity, a bladder, a rectum, a vagina, a nose enter solution of medicinal substance.

In medical practice, especially at treatment of diseases of bronchopulmonary system, gains distribution so-called interstitial E. Thus after administration of medicinal substance carry out by one of the standard ways (intravenously, subcutaneously, intramusculary, in the inhalation way) galvanization of area to an organism patol. the center at a perpendicular arrangement of electrodes. Time of holding a procedure shall correspond to time of achievement of the maximum concentration of medicinal substance in blood.

At the combined ways of treatment medicinal E. it is possible to carry out along with other physiotherapeutic influence. Treat such combined ways ultrasound — an electrophoresis (elektrofonoforez), the dosed vacuum — an electrophoresis (vacuum electrophoresis), an inductothermy — an electrophoresis (induktotermoelektroforez), magnetic field — an electrophoresis (magnetoelectrophoresis), etc. Combination medicinal E. with other physiotherapeutic influences allows to enter into an organism medicinal substance in bigger quantity and deeply, than at one E., also exponentiates its action.

For medical E. apply the pharmaceuticals relating to the most various groups. To us use mestnoanesteziruyushchy means, vitamin, fermental drugs, chemotherapeutic, vasodilating and vasoconstrictors, sedatives, natural compounds more often, etc. The medicinal substances intended for electrophoretic introduction shall be pure, not contain the filling and binding connections, whenever possible their solutions should be prepared just before use. As solvent at preparation of solutions for medicinal E. it is the best of all to use a distilled water. At bad solubility of medicinal substance in water as solvent it is possible to apply alcohol, Dimexidum and other polar solvents allowed GF. Preparation of pharmaceuticals on isotonic solution of sodium of chloride and other solutions electrolytes (see) is undesirable since it sharply reduces introduction to an organism of a medicinal ion. At E. enzymes as solvents use buffered solutions (see).

Dose medicinal E. as well as galvanization: on duration of the procedure from 10 to 30 min. and density of current 0,03 — 0,08 ma/cm 2 . For children and elderly people dosimetric parameters reduce depending on age by 25 — 30%. Appoint from 10 — 12 to 15 — 20 procedures to a course of treatment, to-rye carry out daily or every other day.

For medicinal E. use various devices. Sources of a galvanic current (see. Galvanization ) and impulse diadynamic currents the device Flow-1, AGN-32, AGP-33, SNIM-1 Model-717, the Tone-1 and the Tone-2, the harmonic modulated currents — the devices Amplipuls-ZT are. An amplipulse-4, fluctuating currents — the device ASB-2.



Bibliography: Babsky V. G., Zhukov M. Yu. and Yudovich V. I. The mathematical theory of an electrophoresis, Use to methods of fractionation of biopolymers, Kiev, 1983; Gaal E., Medyeshi G. and Veretsksi of X. An electrophoresis in division of biological macromolecules, the lane with English, M., 198 * Osterman L. A. Methods of a research of proteins and nucleic acids, Electrophoresis and ultracentrifuging, 1981; Parfyonov A. P. Elestroforez of medicinal substances, L., 1973; Ulashchik V. S. Theory and practice of a medicinal electrophoresis, Minsk, 1976, bibliogr.; it, Fizikofarmakologichesky methods of treatment and prevention, Minsk, 1979; Cell electroptoresis in cancer and other clinical reearch, ed. by A. W. Preece a. P. Light, Amsterdam, 1981; Dunn M. Affinity electrophoresis, Lab. Pract., 33, p. 13, 1984; Electrophoresis ’83, Advanced methods biochemical and clinical applications, ed. by H. Hlrai, B. — N. Y., 1984.

E. V. Ramensky; V. S. Ulashchik (fizioter.).

Яндекс.Метрика