Articles
`
`NAGIN K. PATEL AND LLOYD G. NEWSHAM*
`
`EXPERIMENTS IN PHYSICAL PHARMACY. VI.
`
`FACTORS INFLUENCING ERYTHROCYTE FRAGILITY
`
`AND ISOTONICITY DETERMINATION
`
`Early interest in drug solutions having
`the same osmotic pressure as the body
`fluids was relegated to parenteral solu(cid:173)
`tions, but during the past two decades,
`considerable attention has been given
`to the o:motic pressure of collyriums
`and of nasal preparations.
`Isotonic
`solutions are important because they
`cause no swelling or contraction of the
`tissues with which they come in contact
`and produce no discomfort when in(cid:173)
`stilled into the eye, the nasal tract, the
`blood stream, or other body tissue.
`Hypotonic solutions may be rendered
`isotonic by increasing the drug content
`or by
`the addition of some physi(cid:173)
`ologically inert solute. The proportion
`of the solute to be added may be de(cid:173)
`termined experimentally or calculated
`mathematically.
`Students are familiar with the princi(cid:173)
`ple of the plasmolysis experiment from
`
`their biology courses. Drug solutions
`which come in contact with erythrocytes
`should have the same osmotic pressure
`as that of the cell content in order to
`maintain the cell volume or the normal
`tone of the cell. If red cells are · placed
`in water or in sodium chloride solutiom
`containing less than 0.9 per cent sodium
`chloride, water passes into the blood
`cells with a resultant increase in cell
`volume. If the pressure inside the cells
`is sufficiently great, the cells burst. Vari(cid:173)
`ous factors influence the change in the
`cell volume, and students must be
`familiar with these.
`* At the time this experiment was designed,
`Patel was Associate Professor of Pharma(cid:173)
`ceutics, Faculty of Pharmacy and Phar(cid:173)
`maceutical Sciences, University of Alberta;
`present address: Research Laboratories,
`Frank W. Horner Ltd., Montreal, Quebec,
`Canada. Newsham
`is Associate Professor
`of Physiology, Faculty of Medicine, Univer(cid:173)
`sity of Alberta, Edmonton, Alberta, Canada.
`
`1
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`American Journal of Pharmaceutical Education
`
`This paper presents experiments de(cid:173)
`scribing the factors influencing erythro(cid:173)
`cyte fragility, a quantitati,e study on
`osmotic fragility of erythrocytes, and a
`freezing-point-depression (FPD) method
`to determine isotonicity of given solu(cid:173)
`tions.
`
`Theory
`Hemolysis. Only solutes which can(cid:173)
`not pass through a barrier permeable
`to the solvent can exert an osmotic
`pressure ( 1); substances which can pass
`through
`the cell membrane3 cannot,
`therefore, counterbalance
`the osmotic
`pressure exerted by nondiffusible intra(cid:173)
`cellular solutes. Thus, the osmotic con(cid:173)
`centration, measured by a physical
`method based on one of the colligative
`properties, is an expression of the os(cid:173)
`motic pressure only when all the solutes
`present in solution are nondiffusible
`through the cell membrane.
`The membrane of the erythrocyte is
`impermeable, or relatively so, to certain
`solutes. Hence, the volume of water in
`the cell is determined osmotically by the
`composition of the surrounding solu(cid:173)
`tion. The cell can swell or shrink in a
`reversible manner within limits, but at
`a critical volume, the tension on the
`membrane causes it to "rupture" ( or
`become more permeable), and
`the
`cell's contents are released into the sur(cid:173)
`rounding solution.
`When red blood cells ( or other cells)
`are surrounded by aqueous solutions of
`lower osmotic pressure than that inside
`the cell, there is an inward flow of water,
`and the cell volume is increased until the
`osmotic pressures inside and outside the
`cell are equal. If the membrane is too
`fragile to withstand the increased pres(cid:173)
`sure, it will rupture before the osmotic
`pressures on both sides of the membrane
`are equal. This disruption of the red
`cell membrane accompanied by libera(cid:173)
`tion of hemoglobin into the surrounding
`me:lium is termed hemolysis. The op-
`
`2
`
`po3ite process, i.e., the passage of water
`out of the red cell, results in a shrink(cid:173)
`age or crenation of the cell.
`
`By convention, a solution in which
`red blood cells ( or other cells) main(cid:173)
`tain the same volume which they have
`in plasma or tissue fluid is said to be
`isotonic. If the cell volume increases, the
`external solution is hypotonic, and if it
`decreases the solution is hypertonic. Ex(cid:173)
`amples of solutions which are isotonic
`with many mammalian cells are 0.9 per
`cent NaCl and 5.51 per cent dextrose.
`
`Certain chemicals and surface-active
`agents (2, 3) also cause hemolysis, but
`they do so by different mechanisms.
`Solutes such as urea, ammonium chlor(cid:173)
`ide, boric acid, and alcohol pass freely
`through the cell membrane and bring
`about hemolysis becau~e they do not
`provide any osmotic pressure to balance
`that of cell contents. Other substances
`of pharmaceutical interest which do not
`exert any osmotic pressure are succinic
`dinitrite, antipyrine, aminophylline, pro(cid:173)
`pylene glycol, and sodium phenobarbital
`(2). Surface-active agents such as poly(cid:173)
`sorbate 60 increase the permeability of
`the erythrocyte membrane
`to NaCl
`which normally does not pass through
`the membrane.
`
`FPD. The normal freezing point or
`melting point of a pure solvent is the
`temperature at which
`the solid and
`liquid phases coexist in equilibrium un(cid:173)
`der one atmospheric pressure. The
`vapor pressures of the liquid and the
`solid are equal at freezing point. If a
`is dissolved in the liquid the
`solute
`vapor pressure or the escaping tendency
`of the liquid sohent is lowered below
`that of
`the pure solvent. The
`tem(cid:173)
`perature must drop
`in order
`to re(cid:173)
`establish equilibrium between the liquid
`and the solid. In other words, the freez(cid:173)
`ing point of the pure solvent is lowered
`by dissolving a solute in it. The magni-
`
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`Erythrocyte Fragility and lsotonicity Determination
`
`tude of the FPD is proportional to the
`concentration of the solute ( 4).
`The blood is an aqueous system con(cid:173)
`taining dissolved soh;tes and it would
`have a freezing point lower than that
`of pure water. An aqueous solution of
`the rnme
`NaCl, 0.90 per cent, has
`freezing point as that of blood. Since
`water is the solvent in both the case;,
`the FPD of 0.90 per cent NaCl= FPD
`of blood. A solution in question is said
`to be isotonic if it has the same colli(cid:173)
`gative property (FPD or osmotic pres(cid:173)
`sure) as that of blood and if the ery(cid:173)
`throcyte membrane is impermeable to
`the dissolved solutes.
`
`Apparatus and Materials
`Needed for
`this experiment are a
`Beckmann
`cryoscopic
`apparatus,
`a
`cryoscopic thermometer (Heidenhain),
`a magnifying glass, a centrifuge, a
`colorimeter, a microscope, centrifuge
`tubes in rack, defibrinated or heparin(cid:173)
`ized blood 1
`( canine or bovine), pipets
`(10.0 ml. in 0.1 and 0.01), silver ni(cid:173)
`trate, sodium nitrate, sodium chloride
`solutions (0.20, 0.90, and 1.8 per cent),
`3.6 per cent urea, 0.80 per cent am(cid:173)
`monium chloride, and l.9 per cent boric
`acid.
`
`Experimental Procedures
`The experiment can be performed
`over a two-week period with groups of
`two students. If desired, it can be per(cid:173)
`formed
`in one laboratory period by
`omitting the part on "quantitative study
`of erythrocyte fragility." The latter pro-
`
`1coagulation of blood was prevented by the
`following two procedures. Bovine blood was
`obtained from a local slaughterhouse and
`was collected in a container with glass beads
`and agitated
`immediately. In some cases
`agitation with a brush was helpful in sep(cid:173)
`arating the fibrin. In the case of canine
`blood the container was premoistened with
`four to five drops of 5 per cent heparin solu(cid:173)
`tion. The osmotic effect due to the small
`amount of heparin would be negligible, and
`this procedure is preferred.
`
`in the physical
`
`cedure was followed
`pharmacy laboratory.
`Effect of suspending erythrocytes in
`different concentrations of NaCl solu(cid:173)
`tions. Erythrocytes are suspended in
`water and in hypotonic ( distilled water
`or 0.20 per cent NaCl), in isotonic
`(0.90 per cent NaCl), and in hypertonic
`( 1.8 per cent NaCl) solutions. The salt
`solutions are supplied in the laboratory.
`In each of the four labeled test tubes
`containing 5 ml. of each of the above
`NaCl solutions, 0.1 ml. of the given
`blood is pipetted. Each tube is sealed
`with parafilm 2 and inverted gently about
`three times to ensure complete mixing.
`The mixture is allowed to stand for 30
`minutes. The tubes are held against a
`white background ( filter paper), and
`the appearances of the contents are
`noted. Alternately, the tubes are centri(cid:173)
`fuged3 (in which case centrifuge tubes
`should be used) at ca. 1500 rpm for 10
`minutes and the appearance noted; the
`advantage of this procedure is that it
`hastens the rate of sedimentation. This
`alternate procedure was followed by the
`students in the medical school. Either
`procedure gave satisfactory results.
`Effect of urea, ammonium chloride,
`and surface-active agent on the erythro(cid:173)
`cytes. This experiment illustrates that,
`although certain substances (urea, am(cid:173)
`monium chloride, and surface-active
`agents) cause hemolysis, they do so by
`different mechanisms. Using the pro(cid:173)
`cedure described in the preceding sec(cid:173)
`tion, the erythrocytes are suspended in
`5 ml. each of the following solutions:
`0.80 per cent ammonium chloride, 1.8
`per cent urea, 2.5 ml. of 3.6 per cent
`urea + 2.5 ml. of 1.8 per cent sodium
`chloride, and 5 ml. of 0.90 per cent
`sodium chloride + one drop of 10 per
`cent polysorbate 60. It should be noted
`that the above concentrations of am-
`
`2Parafilm, available from supply houses
`:JThe instructor or technician arranged for
`this.
`
`3
`
`l
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`-
`
`-
`
`-
`
`-1
`
`American Journal of Pharmaceutical Education
`
`monium chloride (0.80 per cent) and
`urea ( 1.8 per cent) in the final solu(cid:173)
`tions are iso-osmotic with respect to red
`cells.
`
`Quantitative study of red cell fragility.
`This part describes, in a quantitative
`manner, osmotic hemolysis of the red
`cells in sodium chloride solution of vary(cid:173)
`ing concentrations.
`Ten milliliters of a series of the fol(cid:173)
`lowing concentrations of sodium chlor(cid:173)
`ide are prepared in eight test tubes by
`mixing appropriate amounts of 0.90 per
`cent sodium chloride solution and dis(cid:173)
`tilled water: 0.18, 0.36, 0.45, 0.54,
`0.63, 0.72, and 0.90 per cent. To each
`of these tubes is added (by means of
`a pipet) 0.05 ml. of the blood which
`has been thoroughly mixed ( the red
`cells sediment rapidly, and if the sample
`is not mixed different amounts of red
`cells may be added in each case). The
`tubes are now centrifuged at ca. 1500
`rpm for 10 minutes. By this procedure
`whole red cells and fragments are sedi(cid:173)
`mented. The clear supernatant solution
`is decanted into a cuvette and its optical
`density is measured on a spectrophoto(cid:173)
`meter (Spectronic 20) 4 • The first tube
`containing O per cent NaCl ( distilled
`water) is included as a reference tube,
`and the hemolysis of the red cells in
`this tube is taken as complete or 100 per
`cent for the purpose of calculations.
`Treatment of data. From the optical
`density reading of the first tube showing
`complete hemolysis, the magnitude of
`hemolysis for each of the other tubes is
`given as a simple proportion-e.g., if
`the first tube gives an optical density
`value of 0.600, then a tube giving a
`value of 0.300 yields 0.600/0.300 X
`100 = 50 per cent hemolysis.
`4The instruction sheets covering the principle
`and operation of the
`instrument together
`with its labelled diagram were given to the
`students.
`
`4
`
`A plot is prepared showing per cent
`hemolysis in increasing order along the
`ordinate and sodium chloride solution in
`order of decreasing strength along the
`abscissa. The points are connected in
`a continuous curve. The shape of the
`curve would be expected to be sigmoid
`in character, i.e., convex toward the base
`at lower salt concentrations and convex
`upward as hemolysis nears completion.
`Freezing point determination. The
`is
`Beckmann cryoscopic apparatusG
`portrayed in Fig. 1. In order to reduce
`the rate of cooling, the inner tube, con(cid:173)
`taining the solution, the stirrer, and the
`thermometer 6 , is partially insulated by
`being suspended in a larger test tube
`with an air space in between. The ice(cid:173)
`salt bath temperature should be about
`_50,
`
`STIRRER [stainless
`stee! wire)
`
`OUTER STIRRER
`
`RUBBER STOPPERS
`
`AIR JACKET
`
`Fig. l. The Beckmann cryoscopic apparatus
`
`~•The apparatus shown is of commercial design
`and is ob'.ainable from major supply houses.
`nThe Heidenhain thermometer, with an op(cid:173)
`erating temperature range of + l O to -5 °, is
`very suitable. It has an advantage over the
`Beckmann thermometer in that it is compar(cid:173)
`atively easy to set.
`
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`

`Erythrocyte Fragility and Jsotonicity Determination
`
`About 20 ml. of the liquid under
`study is pipetted into the inner tube. The
`thermometer and the stirrer' are inserted
`with the thermometer carefully mounted
`in such a way that the bulb is about
`halfway between the bottom of the test
`tube and the upper surface of the liquid
`and concentric with the tube so that the
`stirrer can easily pass around it. If the
`thermometer is too close to the bottom
`a bridge of frozen solvent can easily
`form which will conduct heat away from
`the thermometer and result in low read(cid:173)
`ings.
`
`he motion of the stirrer should carry
`
`it from the bottom of the tube up to
`near the surface of the liquid; before
`starting the experiment, it is well to ob(cid:173)
`serve carefully how high the stirrer can
`be raised without too frequent splashing.
`The
`stirring
`should be continuous
`throughout the run and should be at the
`rate of about one stroke per second.
`The ice-salt bath should be stirred a few
`times per minute.
`
`The test tube containing the liquid,
`stopper, thermometer, and stirrer is held
`in a beaker of ice-salt mixture, and the
`liquid is stirred until it visibly starts to
`freeze. The outside of the tube is wiped
`dry and warmed (with stirring) care(cid:173)
`fully with the hand to at least 1 ° above
`the freezing point. Then the tube is
`placed in the assembled apparatus, and
`temperature readings are taken every
`20 ( or 30) seconds with the aid of a
`magnifying glass. The thermometer is
`tapped gently before a reading is taken
`to prevent sticking of the mercury in
`the very fine capillary, and the tem(cid:173)
`perature is estimated in tenths of the
`smallest scale division. Once freezing
`
`7A coiled multiloop stirrer (designed by Wal-
`ter S. Chen and easily prepared from a
`stainless steel wire by wrapping an end of
`the wire around glass tubing) is very effici(cid:173)
`ent and stays in place around the thermome(cid:173)
`ter during stirring.
`
`has occurred, the readings are taken
`for ca. 4-5 minutes; the temperature
`should remain fairly constant during
`that time. Supercooling will probably be
`experienced. A constant
`temperature
`shows that freezing is taking place.
`the
`Using
`the method described,
`freezing points of the following liquids
`are determined: 1) pure distilled water,
`2) 0.9 per cent NaCl, and 3) bovine
`blood or a prescription of 0.5 per cent
`silver nitrate ophthalmic solution ren(cid:173)
`dered isotonic with NaNO3 •
`
`Results and Discussion
`The summary of the results outlined
`is based on the data reported by the
`pharmacy and medical students.
`When
`the erythrocytes were sus(cid:173)
`pended in 0.20 per cent NaCl, hemo(cid:173)
`lysis occurred which was characterized
`by a clear pink solution, the cells having
`burst and released
`the hemoglobin.
`When they were suspended in isotonic
`(0.90 per cent) NaCl and hypertonic
`(1.8 per cent) NaCl, there was an ab(cid:173)
`sence of hemolysis marked by the sedi(cid:173)
`mentation of the cells with a clear zone.
`Microscopic observations showed that
`the red cells retained their normal shape
`in isotonic solution while they were
`crenated in hypertonic solution.
`The suspension of red cells into am(cid:173)
`monium chloride and urea solutions,
`which were
`iso-osmotic with respect
`to the cells, produced hemolysis, indi(cid:173)
`cated by a clear pink coloration of these
`solutions. These solutes pass through the
`membrane of the erythrocytes; therefore,
`they do not contribute to the osmotic
`pressure. The cells in the tube contain(cid:173)
`ing 0.90 per cent NaCl and 1.8 per
`cent urea failed to cause hemolysis, il(cid:173)
`lustrating that urea played no part in
`hemolysis and that the urea solution
`behaved essentially
`like pure water.
`This mixture is then no different from
`0.90 per cent NaCl as far as the erythro(cid:173)
`cytes are concerned. Hemolysis was,
`
`5
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`

`American Journal of Pharmaceutical Education
`
`however, noticed in the tube where the
`cells were suspended in 0.90 per cent
`NaCl
`to which polysorbate 60 was
`added. This
`is because
`the surface(cid:173)
`active agent increases the permeability
`of the erythrocyte membrane to NaCl.
`The student data on the magnitude of
`hemolysis at varying concentrations of
`the salt solutions are plotted in Fig. 2.
`The results gave a S-shaped curve with
`hemolysis appearing
`rather
`sharply.
`There was a range of solute concentra(cid:173)
`tion before hemolysis occurred, indicat(cid:173)
`ing a range of "toughness" of the red
`cell membrane. In certain clinical cases
`of hemolytic anemia ( 5), as depicted in
`Fig. 3, the range of toughness is reduced,
`and osmotic fragility tests will show that
`the erythrocytes of such patients will
`
`100
`
`80
`
`V)
`
`V) >(cid:173)_J
`0
`:E 60
`w
`:r:
`1-z w
`u 40
`0:: w
`a..
`
`20
`
`in concentrations of NaCl
`hemolyze
`which fail to cause rupture of normal
`corpuscles.
`
`PER CENT HEMOLYSIS
`
`• PAMILIAL HEMOLYTIC JAUNDICE
`.t. NORMAL
`• BENZOL POISONING
`♦ COOLEY'S ANEMIA
`
`lO0
`
`80
`
`60
`
`40
`
`20
`
`CONCN. OF SODIUM CHLORIDE ( Gm.I 100 ml,)
`
`Fig. 3. Per cent hemolysis plotted against the
`concentrations of sodium chloride [after Hun(cid:173)
`ter, F.T. (5)]
`
`Based on student group data, typical
`cooling curves of distilled water, bovine
`blood, and isotonic solutions are shown
`in Fig. 4. The dip below the freezing
`point is due to supercooling, which al(cid:173)
`most invariably occurs. In the present
`experiment, supercooling rarely exceeds
`,---.---,,-,,-,----,-17
`
`• DISTILLED WATER l
`
`& BOVINE BLOOD
`■ NaCl, 0.9 Pi:RCl:NT
`0 5 PERCENT•
`1.08 PFRCl:NT
`
`~ -0.20
`
`~ -0.40- -
`w
`"' ::> -0.60
`~
`! 1-
`
`UJ -0.80
`
`- 1.00
`
`0
`040
`080
`CONCN. OF SODIUM CHLORIDE
`(Gm./100 ml.)
`Fig. 2. Student data plotted as per cent hem(cid:173)
`olysis against the concentration of sodium
`chloride. Each point represents the average of
`10 group results.
`
`6
`
`t
`SUPERCOOLING
`
`- I 20
`
`-1.40
`
`Fig. 4. Typical cooling curves of distilled
`water, bovine blood, isotonic sodium chlo(cid:173)
`ride, and an isotonic ophthalmic prescription
`
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`Erythrocyte Fragility and Isotonicity Determination
`
`about 2 ° and is best kept around 1 ° by
`effective stirring. The
`recommended
`procedure for estimating the true freez(cid:173)
`ing point of the solution ( the tempera(cid:173)
`ture at which
`freezing would have
`started in the absence of supercooling)
`is to extrapolate back that point of the
`curve which corresponds to freezing out
`of the solvent until the extrapolate in(cid:173)
`tersects the cooling curve of the liquid
`solution ( 4). To a good approximation,
`the extrapolation may be taken as a
`linear one.
`
`According to the results, the freezing
`point depressions for bovine blood, 0.90
`per cent NaCl, and the silver nitrate
`ophthalmic solution ( a typical prescrip(cid:173)
`tion rendered isotonic) are 0.53 °, 0.54 °,
`and 0.52 °. These values compare favor(cid:173)
`ably with the reported value (3) of
`0.52 °. The results of the entire class
`showing the mean FPD value ±
`the
`standard deviation, and the total number
`of group data points, are summarized as
`follows: 0.90 per cent NaCl, 0.53 ° ±
`0.033 °, 24; bovine blood, 0.52 ° ±
`0.036°, 14; and the prescription, 0.53°
`± 0.041 °, 14, respectively. As is evi(cid:173)
`dent, both the above solutions have the
`same colligative property (FPD) as that
`of blood. Since the erythrocyte mem(cid:173)
`brane is impermeable to the solutes used,
`these solutions would be expected to
`
`have about the same osmotic pressure
`as that of blood, and they are isotonic."
`This experiment demonstrates the quan(cid:173)
`titative determination of isotonicity by
`the freezing point determination. D
`Note added in proof: An osmometer as uti(cid:173)
`lized by W. E. Hall (Am. J. Pharm. Educ., 34,
`204(1970)) would be satisfactory for FPD
`determinations. One of us experimented with
`an Osmette A (Precision Systems) for the
`determination of FPD of isotonic preparations
`and it gave excellent results. The assistance
`of G. J. Weber of Fisher Scientific Co. for a
`demonstration and loan of this apparatus for
`this purpose is appreciated.
`
`References
`( l) Meschia, G. and Setnikar, I., J.
`Gen. Physiol., 42, 429(1918).
`(2) Setnikar, I. and Temelcou, 0., J.
`Am. Pharm. Assoc., Sci. Ed., 48,
`628(1959).
`( 3) Sedam, R. L. and Osol, A., in
`Martin, E. W., Remington's Phar(cid:173)
`maceutical Sciences,
`13th
`ed.,
`Mack Publishing Company, Easton,
`Pennsylvania, 1965.
`( 4) Shoemaker, D. P. and Garland,
`C. W., Experiments in Physical
`Chemistry, McGraw Hill Book
`Company, Inc., New York, 1967,
`pp. 132-140.
`( 5) Hunter, F. T., J. Clin. Invest., 19,
`691 (1940).
`
`7
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