Hemolysis of Erythrocytes in
`Various Iso-osmotic Solutions
`
`By E. R. HAMMARLUNDt and KAJ PEDERSEN-BJERGAARDJ
`
`A study has been made of the degree of hemolysis of human erythrocytes that occurs
`when the red blood cells are incubated in many different pharmaceutically employed
`iso-osmotic solutions. Many distinct differences were noted which further demon-
`strate the frequent dissimilarities between iso-osmotic and isotonic concentra-
`tions. In addition to the hemolytic differences, one of the other main factors i n the
`consideration of the use of isotonic solutions versus iso-osmotic solutions is clearly
`that of the quantities and proportions of the substances used and the resulting rapidity
`and ease of dilution of the medicinal solution by the body fluids.
`
`N A PREVIOUS study of iso-osmotic and isotonic
`Isolutions Hammarlund and Pedersen-Bjergaard
`(1) determined the sodium chloride equivalents
`for 332 pharmaceutical compounds. These were
`calculated from experimentally obtained meas-
`urements of the lowering of the vapor pressure
`and the depression of the freezing point of their
`aqueous solutions. It is well known that solu-
`tions of some medicinally used substances such as
`ammonium chloride, boric acid, ethanol, glycerin,
`and urea fail to prevent the hemolysis of red
`blood cells in iso-osmotic concentrations. For
`such substances that pass through or alter the
`erythrocyte membrane, the iso-osmotic concen-
`tration differs markedly from the isotonic con-
`centration. Husa and co-workers (2-9) have
`investigated the red blood cell hemolytic activity
`of a number of solutions in reference to a de-
`their van’t Hoff i values or
`termination of
`“isotonic coefficients.” However, since the he-
`molytic activities of a large number of pharma-
`ceutical solutions have not been compared
`previously on an equal osmolar basis, except
`those examined by Husa (2-9) and by Setnikar
`and Temelcou (lo), this study was undertaken.
`The purpose was to determine the degree of
`hemolysis which results when deiibrinated blood
`is added to solutions in iso-osmotic concentration
`for 161 substances from our previous study.
`The items selected were those which are com-
`pletely soluble to the extent of their iso-osmotic
`concentrations .
`
`METHOD
`
`The method used is essentially the same as that
`employed by Husa and co-workers (2-9) and is a
`Received March 28 1960 from the School of Pharmacy,
`Washington State Uni;ersity: Pullman.
`Accepted for publication May 18, 1960.
`t Recipient of 1958-1959 Gustavus A. Pfeiffer Memorial
`Research Fellowshp. Present address: College of Phar-
`macy, University of Washington, Seattle.
`t Apoteker Ph.D. Valby Apotek Copenhagen Denmark.
`The autho& wish io thank Dr. &end Aage Schou, Royal
`Danish School of Pharmacy, in whose laboratory this study
`was carried out in 1958-1959, and Dr. E. Freisleben and Dr.
`Else Knudsen of Rigshospital Blood Bank, Copenhagen, for
`generously supplying the numerous small fresh blood samples
`used for this study.
`
`modification of the method by Hunter (11). The
`principal deviations from Husa’s method were that
`iso-osmotic concentrations were employed and an
`aqueous saponin purum,‘ 100 mg./L., was used
`as the 100yo hemolyzing solution €or the erythro-
`cytes instead of 0.1% sodium carbonate solution.
`Finholt (12) and others used a saponin solution for
`a standard since the alkalinity of the carbonate
`solution produces a darker red hemolyzed solution
`which results in a higher colorimetric reading than
`would be obtained a t the body pH of approximately
`7.4. The color given by the hemolysis was de-
`termined as oxyhemoglobin and this was done as
`rapidly as possible following the incubation of the
`defibrinated blood in the solutions being tested.
`Fresh venous blood from human volunteers was
`drawn into a flask containing glass beads. It was
`immediately defibrinated by rotating the flask with
`the beads until the ,fibrin separated. The blood
`was poured into a sniall flask and aerated by rotating
`gently for five minutes. The blood was shaken
`gently by swirling immediately before each 0.1-ml.
`sample was withdrawn with a volumetric pipet.
`The blood was preserved under refrigeration and
`was not used if it had been drawn more than
`twenty-four hours earlier.
`The solutions were freshly prepared with distilled
`water; all chemicals used in the study were identical
`in purity to those reported in the previous study (1).
`Quantitative Determination of the Per Cent He-
`mo1ysis.-Ten milliliters of each solution studied
`was added to each of three centrifuge tubes. Three
`tubes containing 10 ml. of 0.97, sodium chloride
`served as the colorimetric blanks. Three tubes
`containing 10 ml. of saponin solution served as the
`reference color for complete hemolysis; the colori-
`metric readings of which were made at the beginning,
`in the middle, and a t the end of the experimental
`determinations. Exactly 0.1 ml. of fresh defibri-
`nated and aerated blood was added to all tubes by
`means of a volumetric pipet. The tubes were
`stoppered and inverted several times. They were
`placed in a water bath at 25 i 1’ for forty-five
`minutes and were immediately centrifuged for five
`minutes at approximately 2,000 r. p. ni. The su-
`pernate was decanted and its absorbance was de-
`termined in an EEL photoelectric colorimcter with
`a standard green filter. The per cent of hemolysis
`was calculated by dividing the absorbance reading
`for each unknown solution investigated by the
`reading obtained from the average complete he-
`___-
`1 Mrrck and Co., Iidhway, N. J.
`
`24
`
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`

`

`Vol. SO, No. 1. January 1961
`
`molysis times 100%. The instrument was previously
`standardized to read zero absorbance for the 0.90/’,
`sodium chloride blank solutions. The test was
`repeated on another day using different blood
`samples and new solutions for each of those sub-
`stances which gave hemolysis. These two sets of
`values were then averaged and are reported iti
`Table I.
`The pH of each experimental solution was de-
`termined prior to the addition of the blood and this
`value to the nearest 0.1 is included in the results in
`Table I. After the 1% blood sample was added,
`the pH changed slightly in the direction of 7.4.
`The amount of change usually was small and
`depended upon the buffer capacity of the solution.
`If an experimental solution was colored before
`the addition of blood, then this same colored solu-
`tion was employed as the blank. A few solutioris
`were too intensely colored for an accurate colori-
`metric determination of hemolysis and for these
`the method was modified as follows: after centri-
`fuging down the erythrocytes and ghosts,
`the
`colored supernate was decanted;
`the remaining
`erythrocytes were then resuspended in normal saline
`and centrifuged again. The resulting erythrocyte
`volume was compared to the volume in the standard
`0.9% sodium chloride solution for 3n approximate
`percentage of cells hemolyzed.
`I n some instances
`it is possible that the color of the solution and the
`color of oxyhcmoglobin are not exactly additive.
`However, only three solutions in the study (cupric
`sulfate, oxophenarsiiie hydrochloride, and sodium
`ascorbate) were colored appreciably, and no tests
`were made to determine if these colors were addi-
`tive.
`The absorbance readings from some solutions
`resulted in a greater than 100% hemolysis due to
`such €actors as the darkening of the red color in
`alkaline solution and oxidation or reduction reac-
`tions with the blood. These solutions are reported
`as 100% if there were no unhemolyzed erythrocytes
`in the sedinient
`After this study was completed Ansel and Husa
`(13) showed that zinc ions precipitate oxyhemoglo-
`bin; therefore, data for the zinc compounds are not
`included in the study. Furthermore, it is possible
`that other compounds similarly are incompatible
`with oxyhemoglobin since the compatibility of the
`compounds in Table I with oxyhemoglobin was not
`determined. But, any apparent change
`in the
`physical appearance of any of the solutions or of
`the unhemolyzed erythrocytes following incubation is
`reported in the footnotes for Table I.
`
`RESULTS AND DISCUSSION
`
`Hemolytic Values.-The percentage of hemolysis
`found for the 161 compounds studied are listed in
`Table I including the iso-osmotic concentration ( I )
`used for each and the pH of each iso-osmotic solu-
`t ion.
`Solutions of 90 substances prevented heniolysis in
`iso-osmotic concentration and 71 substances showed
`varying degrees from slight to complete hemolysis.
`Solutions of the inorganic salts of moderate pH
`prevented hemolysis of erythrocytes. The car-
`bohydrates and most alkali salts of organic acids
`also prevented hemolysis. Most inorganic and
`
`25
`
`organic acids and alkalies with more extreme pH
`values or with marked oxidation or reduction prop-
`erties usually penetrated or altered the erythrocyte
`sufficiently to give some degree of hemolysis.
`Many arnine salts usually of a monovalent anion
`failed to prevent hemolysis. This is in agreement
`with the results of the permeability of erythrocytes
`to weak electrolytes obtained by Jacobs and
`Stewart (14), Jacobs, Glassman, and Parpart (15),
`and Davson (16), which is summarized well by
`Thomasson and Husa ( 7 ) and Marcus and Husa (9).
`From an examination of the hemolytic data from
`the many compounds studied in equal osmolar
`concentrations, it is evident that there is no distinct
`pattern indicating the predominance of any single
`main mechanism of penetration of the erythrocyte
`by the solute and solvent which results in hemolysis
`of the erythrocyte. T t appears to be a combination
`of factors such as pH, lipoid solubility, the molec-
`ular and ionic size of the particles, and the inhi-
`bition of cholinesterase in the cell membrane (Q),
`to name a few. There are also other complex forces
`which may also play a role because of their possible
`denaturing actions on the plasma membrane pro-
`teins: substances such as alcohols and urea are
`known to rupture hydrogen bonding; low surface
`tension forces may denature proteins; and acids,
`alkalies, and oxidizing agents often change the
`structure of proteins owiug, in part, to their effects
`on benzene ring structures, such as those present
`in the amino acids tyrosine and histidine. The
`picture is still incomplete.
`is well known that
`Ammonium Compounds.-It
`the ammonium salts are an exception to the generally
`accepted view that iso-osmotic solutions of inorganic
`salts of monovalent anions are osmotically indifferent
`to red blood cells.
`The results of the several ammonium compounds
`in this study are presented in Table I1 for compara-
`tive study. The results of ammonium compounds
`from Cadwallader and Husa (6) are also included.
`The erythrocyte membrane is permeable to the
`anions in the first column (about 100~o) and im-
`permeable to those in the second column (0%).
`Since ammonium sulfate and phosphate protected
`erythrocytes from hemolysis, Jacobs and Stewart’s
`(14) statement that ammonium salts of weak acids
`and strong acids will cause hemolysis should be
`amended to include only strong acids of mono-
`valent anions.
`I t is only necessary for a membrane
`to be impermeable to a single species of ion, i. e.,
`anion or cation, for it to be impermeable to the salt
`of which this ion is a part (16).
`Ammonium Chloride.-Since ammonium chloride
`solution is occasionally employed as an i. m. or i. v.
`injection or infusion for the prevention of alkalosis,
`special mention is made of its hemolytic activity-.
`Figure 1 shows a graph of the degree of hemolysis
`of erythrocytes in various concentrations of am-
`monium chloride solutions.
`Instead of obtaining
`the characteristic (17) S-shaped curve (sigmoid)
`as in the case of sodium chloride solutions, one ob-
`tains a radically different curve, Fig. 1. As the
`concentration of ammonium chloride increases, the
`degree of hemolysis decreases from 93% until it
`reaches a minimum point between 4 and 50/,
`ammonium chloride concentration. Four per cent
`ammonium chloride solution was found to require
`
`Novo Nordisk Ex. 2033, P. 2
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`

`

`26
`
`Journal of Pharmaceutical Sciences
`TABLE I.-PERCENTAGE HEMOLYSIS OF ERYTHROCYTES
`IN ISO-OSMOTIC SOLUTIONS
`
`Iso-
`osmotic
`Concen- Hemoly-
`tration,
`sis,
`%
`%
`3.85
`0
`1.39
`100
`1.28
`100
`6.35
`24a
`4.42
`2
`
`Approx.
`PH
`9.2
`6.0
`6 . 1
`3.4
`4 . 4
`
`1.29
`
`0.80
`2.76
`1.30
`
`1.76
`1.68
`
`3.60
`2.64
`
`3.47
`
`4.23
`5.74
`4.98
`6.81
`
`3.96
`3.88
`5.04
`8.85
`3.12
`1.90
`4.56
`
`3.92
`
`97
`
`93
`98
`91
`
`0
`0
`
`0
`98
`
`0
`
`0
`84
`100
`100
`
`1006
`41
`100"
`0
`0
`100
`93
`
`0
`
`7.7
`
`5.0
`5 . 9
`5 . 3
`
`7.9
`5 . 3
`
`9.3
`5 . 7
`
`4.5
`
`5 . 9
`6.8
`5 . 6
`6 . 1
`
`12.0
`4.4
`2 . 2
`5 . 0
`9 . 8
`4 . 6
`6.3
`
`7 . 0
`
`Iso-
`osmotic
`Concen- Hemolv-
`tration.
`sis,
`7%
`%
`
`Approx.
`PH
`
`100"
`
`38
`5
`0
`0"
`0
`100
`95
`0
`0
`0
`100"
`79'
`92
`
`92
`85
`
`100
`100
`1ooc
`0"
`0
`0
`011
`
`0
`98
`
`0
`0
`
`5.70
`
`6.18
`3.32
`
`3.34
`5.05
`4.92
`2.60
`10.87
`1.47
`4.99
`3.30
`1.91
`2.24
`5.67
`
`3 . 7 1
`4.97
`
`9.58
`4.35
`2.30
`9.75
`2.02
`6.30
`5.07
`
`5.07
`4.80
`
`4 74
`3.77
`
`3 . 2 1
`8.59
`
`3 . 4
`
`4 . 7
`4 . 2
`
`8.7
`5.9
`5.9
`5 . 9
`5 . 9
`4 . 7
`6 . 0
`4 . 7
`1.6
`3 . 7
`5.0
`
`5.0
`5.0
`
`9.4
`7 . 1
`2 . 1
`5.8
`6.3
`6.2
`6 . 2
`
`5.3
`5.0
`
`4.5
`5.0
`
`4.5
`5 . 0
`
`Substance
`Acetazoleamide sodium
`Alcohol U. S. P.
`Alcohol, dehydrated N. F.
`Alum (potassium) N. F.
`Amiodoxyl benzoate
`Ammonium carbonate
`u. s. P.
`Ammonium chloride
`u. s. P.
`Ammonium lactate
`Ammonium nitrate
`Ammonium phosphate ,
`dibasic
`Ammoniuni sulfate
`Anlobarbital sodium
`u. s. P.
`&Amphetamine HCI
`Amphetamine phosphate
`N. F.
`Amphetamine sulfate
`u. s. P.
`Amydricaine HC1
`Amylcaine HCl
`Antipyrine N. F.
`2-Methylamino-6-hydroxy-
`6-methylheptane
`(Aranthol )
`Arecoline HBr N. F.
`Ascorbic acid U. S. P.
`Atropine sulfate U. S. P.
`Barbital sodium
`Boric acid U. S. P.
`Butethamine formate
`Caffeine and sodium
`benzoate U. S. P.
`Caffeine and sodium
`salicylate U. S. P.
`Calcium chloride U. S. P.
`Calcium chloride (6H20)
`Calcium chloride,
`anhydrous
`Calcium lactate N. F.
`Calcium pantothenate
`u. s. P.
`Chloramine-T N. F.
`Citric acid U. S. P.
`Cocaine HCl U. S. P.
`Codeine phosphate
`u. s. P.
`p-Propylaminobenzoic
`acid- y-dimethylamino-
`B-oxypropyl ester HCI
`( Cornecaine)
`Cupric sulfate N. F.
`Cupric sulfate, anhydrous
`Cyclopentamine HCl
`Decamethonium bromide
`Dextroamphetamine
`phosphate N. F.
`Dextroamphetamine
`sulfate U. S. P.
`Dextrcse U. S. P.
`Dextrose, anhydrous
`Diethylcarbamazine
`citrate
`Dihydrostreptomycin
`sulfate U. S. P.
`Dipyrone
`Edrophonium chloride
`Ephedrine HCl N. F.
`Ephedrine sulfate U. S. P.
`
`5.77
`1.70
`2.50
`
`1.30
`4.50
`
`5.50
`4.10
`5.52
`6.33
`
`7.29
`
`7.30
`6.85
`4.09
`2.68
`5.00
`
`3.60
`
`4.20
`5.51
`5.05
`
`0
`0
`0
`
`0
`0
`
`0
`100"
`100C
`47
`
`0
`
`100
`traced
`traced
`100
`0
`
`0
`
`0
`0
`0
`
`6.29
`
`100"
`
`19.40
`4.65
`3.36
`3.20
`4.54
`
`0
`0
`0
`96
`0
`
`6.8
`5 . 6
`5 . 7
`
`5.6
`6.7
`
`7.4
`9 . 1
`1 . 8
`4 . 4
`
`4 . 4
`
`6.0
`3 . 9
`4.0
`5.7
`5.7
`
`4 . 7
`
`5.9
`5.9
`6.0
`3 . 7
`
`6 . 1
`7.3
`4.5
`5.9
`5.7
`
`~
`
`~~~~~
`
`Substance
`Epinephrine bitartrate
`Ti. S. P.
`Ethylmorphine HCl
`u. s. P.
`Ethylnorepinephrine H CI
`Fluorescein sodium
`u. s. P.
`&Fructose
`Galactose
`Glycerin U. S. P.
`Glyph ylline
`Guanidine HCl
`Hexamethonium bromide
`Hexamethonium chloride
`Histalog
`Histamine di-HC1
`Homatropine HBr LJ. S. P.
`Hydroxykphetamine
`HBr U. S. P.
`Intracaine HCl
`Iodophthalein sodium
`u. s. P.
`Isoniazid U. S. P.
`Lactic acid U. S. P.
`Lactose U. S. P.
`Magnesium chloride
`Magnesium sulfate
`Mannitol N. F.
`Menadione sodium
`bisulfite U. S. P.
`Meperidine HCl U. S. P.
`Mephentermine sulfate
`u. s. P.
`Methacholine bromide
`Mcthacholine chloride
`u. s. P.
`Methadone HCl U. S. P.
`Methamphetamine HC1
`u. s. P.
`Methenamine U. S. P.
`Methoxamine HCl U. S. P.
`Methvlatrouine bromide
`Mondethanblamine N. F.
`Naphazoline HCl N. F.
`Neoarsphenamine
`Nicotinamide U. S. P.
`Nikethamide U. S. P.
`Oxophenarsine HC1
`u. s. P.
`Penicillin G, potassium
`u. s. P.
`Pentylenetetrazole U. S. P.
`Phenobarbital sodium
`U. s. P.
`Phenol U. S. P.
`Phenylephrine HCl
`u. s. P.
`Phenylpropanolamine HCl
`Phenylprop ylmethylamine
`HC1
`Pilocarpine HCl U. S. P.
`Potassium chlorate N. F.
`Potassium chloride U. S. P.
`Potassium iodide U. S. P.
`Potassium nitrate N. F.
`Potassium phosphate N. F.
`Potassium uhoso h at e .
`monobasic
`Potassium sulfate
`ProbarbitalsodiumN. F
`Procaine HCl U. S. P.
`
`~
`
`~~
`
`0
`1ooc
`
`97
`100
`88
`0
`100
`100
`17
`100
`100
`tracea
`
`0
`100
`
`0
`o h
`
`0
`95
`
`95
`89
`0
`0
`0
`0
`0
`0
`0
`0
`91
`
`2.75
`3.68
`3.82
`7.03
`1.76
`3.99
`2.32
`4.49
`5.94
`
`3.67
`
`5.48
`4 . 9 1
`
`3.9E
`2.80
`
`3.00
`2.60
`
`2.70
`4.08
`1.88
`1.19
`2.59
`1.62
`2.08
`~-
`2.18
`2 . 1 1
`3.10
`5.05
`
`~
`
`5.9
`8.4
`5.2
`5.7
`11.4
`5.3
`7.8
`7.0
`6.9
`
`2.3
`
`6.2
`6.7
`
`9 . 2
`5.6
`
`4.5
`5.3
`
`5.4
`4.0
`6.9
`5.9
`7.0
`5.9
`8 . 4
`
`4.4
`6.6
`10.0
`5.6
`
`Novo Nordisk Ex. 2033, P. 3
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`
`

`

`Vol. 50, No. 1, January 1961
`
`TABLE I (continued)
`
`27
`
`Iso-
`osmotic
`Concen- Hemoly- Approx.
`tration
`sis
`%
`7 0
`
`PH
`
`2.23
`
`4.45
`1.47
`2.53
`3.95
`1.61
`2.98
`5.48
`
`4.48
`9.25
`
`3.85
`4.24
`4.82
`3.90
`3.17
`
`2.67
`4.24
`3.05
`4.22
`4.92
`
`3.40
`1.63
`2.93
`3.55
`
`0
`
`0
`0
`0
`0
`0
`0
`0
`
`85"
`0
`
`0
`0
`0
`75'
`
`0
`
`0
`87'
`93
`100
`75
`
`0
`100
`100
`0
`
`9.2
`
`9.2
`7.8
`6.7
`6 . 1
`6.2
`7 . 4
`5 . 9
`
`3.5
`6.4
`
`8.7
`9.5
`9.9
`1.7
`5.7
`
`4.7
`3.0
`4.8
`6.0
`5 7
`
`5.9
`6.6
`6.3
`9.7
`
`Substance
`Sodium phosphate,
`dibasic (2Hz0)
`Sodium phosphate,
`dibasic (12H20)
`Sodium propionate N. F.
`Sodium salicylate U. S. P.
`Sodium sulfate N. F.
`Sodium sulfate. anhvdrous
`Sodium thiosulfate N. F.
`Sorbitol ( l / 2 H20)
`u. s. P.
`Succinylcholine chloride
`Sucrose U. S. P
`u . s. P.
`Sulfacetamide sodium
`Sulfadiazine sodium
`u. s. P.
`Sulfathiazole sodium N. F.
`Tartaric acid N. F.
`Tetraethylammonium
`bromide
`Tetraethylammonium
`chloride
`Thiamine HC1 U. S. P.
`Tolazoline HC1 U. S. P.
`Trimethadione U. S. P.
`Tropacocaine HC1
`Tuaminoheptane sulfate
`N. F.
`Urea U. S. P.
`Urethan U. S. P.
`Vinbarbital sodium
`
`Substance
`Propylene glycol U. S. P.
`QujyiZe and urea HC1
`IY. r.
`Racephedrine HC1 N. F.
`Resorcinol U. S. P.
`Scopolamine HBr U. S. P.
`Silver nitrate U. S. P.
`Silver protein, mild N. F.
`Sodium acetate, anhydrous
`U. s. P.
`Sodium aminosalicylate
`Sodium arsenate, dibasic
`Sodium ascorbate
`Sodium benzoate U. S. P.
`Sodium bicarbonate
`Sodium biphosphate,
`anhvdrous
`Sodium biphosphate
`u. s. P.
`Sodium biphosphate
`(2H20)
`Sodium bisulfite U. S. P.
`Sodium borate U. S. P.
`Sodium cacodylate N. F.
`Sodium carbonate,
`anhydrous
`Sodium carbonate, mono
`hydrated U. S. P.
`Sodium chloride U. S. P.
`Sodium citrate U. S. P.
`Sodium iodide U. S. P.
`Sodium lactate
`Sodium metabisulfite
`Sodium nitrate
`Sodium nitrite U. S. P.
`Sodium phosphate,
`exsiccated, N. F.
`Sodium phosphate N. F.
`
`Iso-
`osmotic
`Concen- Hemoly-
`tration,
`SIS.
`%
`%
`2.00
`100
`641
`4.50
`3.07
`94
`3.30
`96
`8
`7.85
`2.74
`O d
`5.51
`0
`1.18
`0
`
`3.27
`3.83
`3.00
`2.25
`1.39
`2.10
`2.45
`
`2.77
`1.50
`2.60
`3.30
`1.32
`
`1.56
`0.90
`3.02
`2.37
`1.72
`1.38
`1.36
`1.08
`
`1.75
`3.33
`
`0
`0
`0
`0
`0
`
`0
`
`0
`
`0
`0;
`0
`0
`
`100
`
`100
`0
`0
`0
`0
`5i
`0
`0"
`
`0
`0
`
`Approx.
`PH
`5 . 5
`
`2 . 9
`5.7
`5.0
`4.8
`5.0
`9 . 0
`8.1
`
`7 . 3
`8 . 8
`6 . 9
`7.5
`8.3
`4.1
`4 . 1
`
`4 . 0
`3.0
`9.2
`8.0
`
`11.1
`
`11.1
`6.7
`7.8
`6.9
`6.5
`4.5
`6.0
`8 . 5
`
`9.1
`9.2
`
`(I R. B. cells turned black color. b Solution turned olive-
`green color.
`R. B.
`c Solution turned brown-black color.
`cells shrunk in size and turned black color. eR. B. cells
`f Solution and R. B. cells darkened.
`clumped.
`0 Solution
`became slightly turbid. h R . B. cells turned brown color
`and solution became milky.
`i R. B. cells turned violet
`color. 3 Solution and R. B. cells turned violet color.
`
`the presence of 2.5y0 dextrose to prevent hemolysis.
`The degree of hemolysis is then greater in the
`ammonium chloride solutions from this minimum
`point up to 10%.
`Various proportions of sodium chloride were
`added
`to
`three different
`concentrations of
`ammonium chloride solution: 0.8, 0.6, and 0.4%!.
`It was found that an approximately identical pro-
`portion of sodium chloride 0.65y0, was required to
`prevent hemolysis of erythrocytes by each of the
`ammonium chloride solutions, Table 111.
`The commonly accepted iso-osmotic composition
`of 0.8% ammonium chloride gives 93% hemolysis.
`To prevent hemolysis of erythrocytes, it was found
`under the conditions of the procedure mentioned
`previously that 0.65% sodium chloride or 4% dex-
`trose must be added to the 0.8% ammonium chlo-
`ride solution. This will give an isotonic but hyper-
`osmotic solution.
`hemolysis ob-
`Amphetamine Compounds.-The
`tained in solutions of
`the amphetamine salts of
`monovalent and polyvalent anions provides an inter-
`esting comparison with the ammonium salts. Iso-
`osmotic solutions of amphetamine hydrochloride,
`hydrobromide, and methamphetamine hydrochloride
`
`each produced about 100% hemolysis while the
`solutions of amphetamine phosphate and sulfate
`prevented hemolysis. The erythrocytes are more
`permeable to the monovalent anions of ampheta-
`mine than to the polyvalent sulfate and phosphate
`anions. This is in agreement with Davson (16) and
`Thomasson and Husa (7) who found that the sul-
`fate and other polyvalent anions penetrate the
`erythrocyte very slowly in comparison with uni-
`valent anions.
`and Husa
`Ephedrine Compounds.-Thomasson
`(7) mentioned the difference in the hemolytic ac-
`tivity between ephedrine hydrochloride and sulfate
`solutions.
`In
`the present study racephedrine
`hydrochloride was also found to allow nearly the
`same degree of hemolysis as ephedrine hyprochloride
`94 and loo%, whereas ephedrine sulfate prevented
`hemolysis. This is, one would assume, because the
`membrane is more permeable to the chloride than
`to the sulfate ion.
`Because of the great difference in their hemolytic
`effects, solutions of ephedrine hydrochloride and
`sulfate were investigated further in order to find out
`how much sodium chloride and sodium sulfate must
`be added to prevent the hemolysis of their iso-
`
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`

`28
`TABLE II.-ERYTHROCYTE HEMOLYSIS IN
`Iso-
`OSMOTIC SOLUTIONS OF VARIOUS AMMONIUM SALTS
`
`Journal of Pharmaceutical Sciences
`TABLE III.-ERYTHROCYTE HEMOLYSIS IN AM-
`MONIUM CHLORIDE SOLUTIONS CONTAINING VARY-
`
`ING AMOUNTS OF SODIUM CHLORIDE -- Hemolysis, Yo--
`
`NaCl
`Added, Yo
`0.50
`0.55
`0.60
`0.65
`0.70
`0.80
`
`Ammonium Chloride Concentration
`0.6%
`0.8%
`0.4%
`80
`76
`82
`39
`45
`43
`3
`6
`2
`0
`0
`0
`0
`0
`0
`0
`0
`0
`
`HEMOLYSIS IN
`TABLE IV.-ERYTHROCYTE
`ISO-
`OSMOTIC EPHEDRINE HYDROCHLORIDE SOLUTIONS
`(3.27,) CONTAINING VARYING PROPORTIONS OF
`SODIUM CHLORIDE
`
`0
`-
`
`NaCl
`Added, %
`0 . 3
`0 . 4
`0 . 5
`0.6
`0.7
`
`Hemolysis, yo
`94
`91
`6
`0
`0
`
`He-
`moly-
`SlS,
`%
`
`0
`
`0
`
`0
`
`0
`
`0
`
`Substance
`Ammonium
`acetatea
`Ammonium
`benzoate"
`
`Ammonium
`carbonate
`Ammonium
`chloride
`Ammonium
`lactate
`Ammonium
`nitrate
`
`Ammonium
`salicylate"
`
`He-
`moly-
`sis,
`%
`
`100
`
`100
`
`98
`93
`
`98
`92
`
`100
`
`Substance
`Ammonium
`citrate"
`Ammonium
`phosphate,
`dibasic
`Ammonium
`sulfate
`Ammonium
`tartrate"
`Tetraethyl-
`ammonium
`bromide
`Tetraethyl-
`ammonium
`chloride
`
`a Results from Cadwallader and Husa (6)
`
`60 -
`
`9 5 0 -
`2. e
`f 40-
`0 5 30-
`
`TABLE V.-ERYTHROCYTE HEMOLYSIS
`IN VARIOUS
`
`EPHEDRINE HYDROCHLORIDE SOLUTIONS MADE Iso-
`OSMOTIC WITH SODIUM CHLORIDE
`
`Ephedrine HC1
`Iso-
`osmotic
`Concn.,
`%
`Multiple
`4.0
`1 '/4
`3.2
`1
`2.4
`3/4
`1.6
`'/2
`1.2
`"8
`0.8
`I/<
`
`---NaCl
`Iso-
`osmotic
`Multiple
`0
`0
`1/4
`'/2
`/ 8
`3 1 4
`
`Added-
`Concn.,
`%
`0
`0
`0.23
`0.45
`0.56
`0.68
`
`He-
`mql y-
`SlS,
`%
`99
`96
`96
`94
`1
`0
`
`TABLE V1.-ERYTHROCYTE HEMOLYSIS IN VARIOUS
`SOLUTIONS MADE Iso-
`EPHEDRINE HYDROCHLORIDE
`OSMOTIC WITH SODIUM SULFATE
`
`Ephedrine HC1
`Iso-
`osmotic
`Concn.,
`%
`Multiple
`1
`3 . 2
`2.4
`"4
`1.6
`'/2
`0 . 8
`'/4
`
`--NazSO,
`Iso-
`osmotic
`Multiple
`0
`'/4
`'/Z
`"4
`
`Added-
`Concn.,
`%
`0
`0.38
`0.76
`1.14
`
`He-
`moly-
`sis,
`%
`96
`16
`0
`0
`
`and the results are presented in Table V for the
`addition of sodium chloride and Table VI for the
`addition of sodium sulfate.
`Ephedrine hydrochloride solutions have strong
`hemolytic activity until their concentrations are
`reduced to less than one-half iso-osmotic value with
`sodium chloride.
`The presence of sodium sulfate depresses much
`more strongly the hemolytic activity of ephedrine
`hydrochloride than does an equivalent quantity of
`sodium chloride.
`The per cent of hemolysis given by various pro-
`portions of the iso-osmotic concentration of ephed-
`rine sulfate solutions adjusted with sodium chlo-
`
`I
`
`00
`
`i
`
`i
`
`i
`i i
`i
`h
`lrnrnO"l"rn Chlo. rJe CoICCP'mllO"
`IV. 1
`Fig. 1.-Erythrocyte
`hemolysis in amnionium
`chloride solutions of varying concentrations.
`
`Q
`
`d
`
`
`
`1;
`
`osmotic solutions and of various dilutions of these.
`Various proportions of sodium chloride were added
`to iso-osmotic solutions of ephedrine hydrochloride
`and the degree of hemolysis was determined,
`Table IV.
`When 0.6% sodium chloride is added, the he-
`molysis is prevented. This is slightly more than the
`required amount of sodiuni chloride which alone
`would retard hemolysis. Thus the ephedrine hy-
`drochloride is similar to ammonium chloride and
`other substances, as reported by Thomasson and
`Husa (7), which act in a negative manner in pre-
`venting hemolysis in the presence of sodium chlo-
`In other words, their iso-osmotic solutions
`ride.
`cause an increased fragility of erythrocytes.
`The per cent of hemolysis given by various pro-
`portions of the iso-osmotic concentration of ephed-
`rine hydrochloride solutions containing the neces-
`sary amounts of sodium chloride or of sodium
`sulfate to maintain iso-osmoticity was determined
`
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`

`29
`
`Ephedrine Sulfate
`Iso-
`osmotic
`Concn.,
`%
`Multiple
`1
`4.54
`3.41
`3/4
`2.27
`1/2
`1.14
`
`lil
`
`Y N a C l Added?
`Iso-
`osmotic
`Concn.,
`%
`Multiple
`0
`0
`0.23
`’/4
`0.45
`‘/z
`.~
`0.68
`314
`
`He-
`moly-
`SlS,
`%
`0
`0
`0
`0
`
`TABLE VIII.-ERYTHROCYTE HEMOLYSIS IN Iso-
`OSMOTIC BORATE: BORIC ACID SOLUTIONS
`
`Vol. 50, No. 1, Janziary 1961
`TABLE VII.-ERYTHROCYTE HEMOLYSIS IN EPHE-
`of hemolysis took place in a rather limited pH
`DRINE SULFATE SOLUTIONS MADE ISO-OSMOTIC
`interval, it can be concluded perhaps that the pH
`WITH SODIUM CHLORIDE
`is of no real influence in this instance.
`Trolle-Lassen (18) has shown previously that
`red blood cells hemolyze only to a very slight extent
`in isotonic solutions of sodium chloride containing
`2% or less of boric acid. But in all cases the
`erythrocytes were completely hemolyzed within a
`few hours if the solutions contained 3y0 or more of
`boric acid.
`Reversed Proportion of Erythrocytes to Total
`Solution Volume.-The
`results in Table I show that
`there is a large number of substances which in iso-
`osmotic concentration fail to prevent hemolysis of
`red blood cells when a small amount ol blood is
`mixed with a large volume of solution; however,
`this proportion of solution to blood and tissue fluids
`(10 ml. solution to 0.1 ml. blood or 10O:l) is
`not very often obtained
`in
`the body and
`hence the dauger from hemolysis usually is much
`less than is indicated by in vitro studies. The
`average proportional volume of a medicinal injec-
`tion usually is too small when compared to the total
`blood volume and adjacent tissue fluid volume to be
`a serious factor in all but exceptional cases.
`The hemolytic activities of a few strongly hemo-
`lytic substances were determined in the completely
`reversed proportion of solution to blood volume
`(1 : 10). One-half milliliter of iso-osmotic concen-
`tration of ammonium chloride, boric acid, ethanol
`sodium carbonate, and urea was added to 10 ml.
`of a 1: 1 mixture of fresh, defibrinated whole blood
`and 0.9% sodium chloride solution. The blood was
`diluted with an equal volume of sodium chloride
`solution for ease in centrifuging and colorimetric
`measuring. Blanks were made containing
`the
`blood with normal saline solution replacing the
`hemolytic solution. The incubation and photo-
`electric determination of the color of the solution
`was made in a similar manner as before and showed
`that there was no hemolysis (07,) of the erythro-
`cytes when mixed in proportion of 10: 1 with each
`of the solutions tested.
`When the volume of urea solution was increased
`to 1 ml. (which resulted in a mixture of one part of
`iso-osmotic urea solution to five parts of blood),
`there was slight hemolysis (10$70). When 2 ml.
`of urea solution (1 part iso-osmotic urea solution to
`2ll2 parts blood) was investigated, a moderate
`degree of hemolysis resulted (40%). This indicates
`that one of the factors in the consideration of the
`use of isotonic solutions uersus iso-osmotic solutions
`is clearly that of the quantity and propot tions of the
`substances used and the resulting rapidity and ease
`of the dilution of the medicinal solution by the
`body fluids.
`
`
`
`Boric Acid
`Solution
`Vol-
`Mole
`ume,
`Frac-
`tion
`ml.
`50
`1.00
`45
`0.98
`40
`0.95
`35
`0.91
`30
`0.87
`25
`0.82
`20
`0.75
`15
`0.66
`0.53
`10
`5
`0.34
`0
`0
`
`
`
`Sodium Borate
`Solution
`Vol-
`Mole-
`ume,
`Frac-
`ml.
`tion
`0
`0
`5
`0.02
`0.05
`10
`15
`0.09
`20
`0.13
`25
`0.18
`30
`0.25
`35
`0.34
`40
`0.47
`45
`0.66
`50
`1.00
`
`He-
`mply-
`SIS. %
`100
`99
`99
`99
`97
`23
`1
`0
`0
`0
`0
`
`PH
`4 . 7
`7 . 1
`7 . 6
`7.9
`8.1
`8 . 4
`8.6
`8 . 8
`-~
`8.9
`9 . 0
`9.2
`
`ride was determined and the results are given in
`Table VIT.
`Ephedrine sulfate solutions prevented hemolysis
`of red blood cells even though there was an ap-
`preciable quantity of chloride present for cellular
`exchange and for maintenance of cellular neu-
`trality.
`iso-osmotic (1.39%) solution of
`Ethanol.-An
`ethanol failed completely to prevent hemolysis of
`erythrocytes. It was found necessary to add 0.5y6
`sodium chloride to the ethanol solution to prevent
`hemolysis. This is the same amount of sodium
`chloride which alone will just prevent hemolysis.
`The ethanol in iso-osmotic concentration, therefore,
`did not increase the fragility of the erythrocytes as
`did the ammonium chloride.
`Erythrocyte Hemolysis in the System : Boric Acid-
`Sodium Borate.-Since
`an
`iso-osmotic solution
`(1.9%) of boric acid fails to prevent hemolysis of
`erythrocytes while an iso-osmotic solution (2.6%)
`of sodium borate prevents hemolysis, a study was
`made to show the degree of erythrocyte hemolysis
`in various proportions of the two iso-osmotic solu-
`tions, Table VIII. The pH of each solution was
`obtained before the addition of the blood sample.
`For comparative purposes the quantities of
`the
`two solutes in the various mixtures are reported as
`mole fractions of the total solute only.
`Since boric acid is more ether soluble than is
`sodium borate, it is undoubtedly more lipoid soluble,
`and therefore it penetrates the plasma membrane
`of the erythrocyte more readily than does the sodium
`borate. The concentration of the reactants in the
`mixture which prevented hemolysis was 0.75 or
`less mole-fractions of boric acid and 0.25 or more
`mole-fractions of sodium borate, and with a pH
`8.6 to 9.2. Since an appreciable change in the degree
`
`SUMMARY
`
`1. The degree of hemolysis of human eryth-
`rocytes was determined in 161 different phar-
`maceutically employed iso-osmotic solutions.
`2. Ninety substances prevented hemolysis
`in iso-osmotic concentration and 71 substances
`showed varying amounts of hemolysis.
`3. Solutions of carbohydrates, most alkali
`
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`

`30
`
`salts of organic acids, and inorganic salts with
`moderate pH prevented hemolysis.
`4. Most inorganic and organic acids and
`alkalies with more extreme pH values or which
`have marked oxidation or reduction properties
`usually penetrated or altered the erythrocyte
`sufficiently to give some degree of hemolysis.
`5.
`Many amine salts usually of a monovalent
`anion failed to prevent hemolysis, whereas amine
`salts of di- and tri-valent anions usually did pre-
`vent hemolysis.
`A hemolytic curve for ammonium chloride
`6.
`solution is p