628
`
`ASSOCIATION Vol. XLVIII, No. 11
`JOURNAL OF THE AMERICAN PHARMACEUTICAL
`Worth, H. M., and Harris, P. N., Antibiotic Ann., 1958/59,
`354. (3) Stephens, V. C., Conine. J. W., and Murphy, H. W.,
`THIS OURNAL 48, 620(1959).
`( 4 j Andersbn. R. C.. Henderson. F. G.. and Chen.
`K . K:, ibid., 32, 204(1943).
`( 5 ) Bollman, J. L.. J . Lab. Clin. 'Wed., 33, 1348(1948).
`(6) Herberg, R. J.. in press.
`(7) Kirshbaum, A,. Bowman, F. W., Wintermere, D.
`M;.,anfi<Friedman. E. R., Antibiotics & Chemotherapy. 3 ,
`33 I ( 1Yb3J.
`( 8 ) Freese, H. B., Hambourger, W. E., Calvin, L. O.,
`and Green, D. M., J . Pharm. Exfill. Therafi., 104, N(1952).
`(9) Griffith, R. S., Stephen, V. C., Wolfe. R. N.. Boniece,
`W. S.. and Lee, C . C.. Antibiotic Med. & Clin. Therafiy, 5 ,
`finwiam)
`_ _ " \ _ " .__,.
`(10) Fitzhugh, 0. G., and Nelson, A. A,, THIS JOURNAL,
`37,29(1948).
`
`partly due to its limited biliary excretion and its
`slow rate of disappearance from the various tis-
`sues.
`9. Part of the blood erythromycin is loosely
`bound to red blood cell component or adsorbed on
`the cells.
`
`REFERENCES
`(1) McCuire, J. M., Bunch, R. L., Anderson, R. C..
`Boaz, H. E., Flynn. E. H . , Powell, H. M . , and Smith, J. W.,
`Anlibiolics & Chemolherafiy, 2,281 (1952).
`(2) Lee, C. C., Anderson, R. C.. Henderson, F. G.,
`
`Osmotic Concentration and Osmotic Pressure in
`Injectable Solutions*
`By I. SETNIKAR a n d OLIMPIA TEMELCOU
`
`A method for determining the osmotic pressure of injectable solutions by measuring
`the variations of the red-cell volume is described. By means of this method, sub-
`stances of pharmaceutical interest can be classified into different groups according
`to their diffusibility through the erythrocyte membrane and their action upon it. It
`was demonstrated in vitro and in vivo that for many substances the iso-osmotic con-
`centration is not equivalent to the isotonic concentration and that the confusion
`between iso-osmia and isotonia can have dangerous consequences.
`
`T IS COMMON KNOWLEDGE that only solutes
`
`I which cannot pass through a barrier per-
`
`meable to the solvent can exert an osmotic pres-
`sure (1); substances which can pass through the
`cell membranes cannot, therefore, counterbalance
`the osmotic pressure exerted by nondiffusible
`intracellular solutes.
`Therefore, the osmotic concentration, measured
`by physical methods based on one of the colliga-
`tive properties, is an expression of the osmotic
`pressure only when all the solutes present in solu-
`tion are nondiffusible through the cell membranes,
`otherwise a solution found to be iso-osmotic is hy-
`potonic for the cells.
`This distinction would be of little practical
`importance were it possible to accept the view of
`Szekely and Goyan (2) to the effect that, of the
`substances in pharmaceutical use, those freely
`diffusible through the cell membranes are excep-
`tional. The researches performed with a hemo-
`lytic method by Husa, et al. (3-S), demonstrate
`that, on the contrary, many substances in com-
`mon pharmaceutical use, at a concentration iso-
`osmotic with blood, cause hemolysis for the very
`reason that they are unable to counterbalance the
`intracellular osmotic pressure.
`While the hemolytic method can demonstrate
`very clearly the difference between solutes which
`are diffusible through the membrane of red cells
`and those which are not, it is not so easy to deter-
`* Received July 6. 1959, from the Research Department,
`Recordati Laboratorio Farmacologico S. p. A., Milano, Italy.
`
`mine the isotonic concentration because, for this
`purpose, it is necessary to start from the premise
`that for all solutes there is a single ratio between
`isotonic concentration and hemolytic concentra-
`tion, whereas it has been shown that, on the con-
`trary, this ratio may vary from 1.4 to 3.1 (9).
`Efforts have therefore been directed to the
`search for a method of direct determination of iso-
`tonic concentration and it has been found that
`this could be done fairly simply by means of a
`suitable modification of the hematocrit method
`used by Eijkman (10). This paper describes the
`method employed and the results obtained there-
`from.
`
`METHOD
`Human blood was drawn from a forearm vein;
`rabbit blood by cardiac puncture. The syringes
`used for drawing the blood were moistened with an
`0.65% (isotonic) solution of NaF containing 5y0
`heparin. The blood samples were centrifuged, the
`separated red cells were added to an equal volume of
`the solution under examination, and this suspension
`was centrifuged for thirty minutes at 3,000 r. p. m.
`in Wintrobe's hematocrit tubes. To determine
`the volume that the red cells would maintain in an
`isotonic solution, a similar test was performed
`mixing the red cells with the plasma of the same
`specimen of blood.
`
`RESULTS
`ratio between the volume of rabbit
`NaC1.-The
`red cells and the concentration of NaCl is given in
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`SCIENTIFIC EDITION
`
`629
`
`Fig. 1. In contact with plasma, red cells maintained
`a volume equal to that which they would take up in
`contact with an 0.93% solution of NaCl. This con-
`centration is, therefore, isotonic with the red cell
`specimen used.
`
`20
`
`30
`
`10
`DEXTROSE, yo
`of dextrose solutions at dif-
`Fig. 2.-Effects
`ferent concentrations upon the volume of the red
`cells of the rabbit and of man. The dotted line
`indicates the isotonic volume. 0-0. human red
`cells; 0-0, rabbit red cells.
`
`0
`
`NaCI. %
`Fig. 1.-Effect of the NaCl concentration upon
`the volume of rabbit red cells. The dotted line
`represents the volume of the cells suspended in
`plasma (isotonic volume). For these red cells the
`isotonic concentration of NaCl was 0.93%.
`
`Urea.-Solutions of urea from 1 to 2.6% (a 1.87,
`solution is iso-osmotic) caused complete hemolysis.
`The addition of NaCl at concentrations from 0.5 to
`0.9% to an iso-osmotic solution of urea prevented
`laking, and the volume of the red cells was equal to
`that determined by solution of NaCl a t the same
`concentration but without urea. Thus, urea does
`not of itself have a hemolytic effect, as in the
`opinion, for example, of Ebina (11);
`laking is
`brought about by the incapacity of this substance
`to counterbalance the intracellular osomotic pres-
`sure.
`In other words, as regards osmotic pressure,
`it is as if urea were not present in solution.
`The incapacity of urea to exert an osmotic pres-
`sure can be demonstrated also in vivo. If 15 cc./Kg.
`of a 1.8% solution of urea is injected intravenously
`into rabbits, extensive hemolysis is observed, due
`to the destruction of about 3% of the red cells. A
`similar phenomenon is observed when one admin-
`isters the same quantity of distilled water. He-
`molysis can be entirely avoided by rendering the
`solution of urea isotonic with a 0.9% solution of
`NaCI.
`Dextrose.-While dextrose exerts an osmotic pres-
`sure equal to its concentration on the erythrocytes of
`rabbits, on human erythrocytes the isotonic concen-
`tration is almost twice the iso-osmotic concentration
`(Fig. 2). The membranes of human erythrocytes
`would therefore seem to be partially permeable to
`dextrose.
`It is interesting to note that the resistance
`of human red cells increases in solutions of dextrose;
`the cell volume in hypotonic solutions can attain
`values practically twice those which usually precede
`hemolysis.
`This increase in cell resistance perhaps ac-
`counts for the results obtained by Grosicki and
`Husa (4), who noted no difference between the
`
`v
`2
`v1 ;
`0.30 2 a w
`0.40 ti L
`0.50 5
`0.60 a
`1.00 g
`N w w
`d 14
`
`I
`
`40
`
`7
`5
`8
`0
`Fig. 3.-Effects of the pH upon the volume of
`red cells and thus also upon intracellular osmotic
`pressure. The cations concentration was maintained
`constant to 155 meq./L. of Na+.
`Left ordinate, cell volume;
`right ordinate,
`depression of the freezing point on a scale so ad-
`justed that, for a solution of NaCI, the depressions
`of the freezing point would correspond to the cell
`volume; abscissa. pH of the buffer-red cell mixture.
`The isotonic cell volume is shown by a dotted
`line. The straight line and the empty signs rep-
`resents the cell volume, the dotted line and the
`full signs give the cryoscopic depression. 0-
`Buffers of acetic acid-sodium acetate, 0- buffers of
`barbital-
`NaH2P04- hTa2HP04, A-buffers of
`sodium barbital. Note the poor correlation be-
`tween the osmotic concentration measured by the
`cryoscopic method and the osmotic pressure exerted
`by the solutions upon the red cells.
`hemolytic concentration of dextrose for human red
`cells and those for rabbit red cells.
`Procaine Hydrochloride.-At
`its iso-osmotic con-
`centration (5.057,), procaine hydrochloride causes
`In contra-
`hemolysis of the red cells of the rabbit.
`distinction to what has been observed with urea, to
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`630
`
`JOURNAL OF THE AMERICAN PHARMACEUTICAL
`ASSOCIATION Vol. XLVIII, NO. 11
`DISCUSSION
`Similar experiments carried out on substances of
`pharmaceutical interest showed that these could be
`classified into the following groups :
`Group 1.-Substances whose iso-osmotic con-
`centration is isotonic: NaCl (0.9%), KCl (1.1976),
`sodium thiosulfate N. F. (2.98y0), sodium borate
`U. S. P. (2.670). sodium propionate N. F. (1.4Trjb),
`sodium benzoate U. S. P. (2.25y0), sodium barbital
`(3.14Oj,), sorbitol (5.48y6), and dextrose U. S. P.
`(5.5. o/; ) for rabbit red cells.
`Group 2.-Substances which do not exert any
`osmotic pressure: urea, succinic dinitrile, anti-
`pyrine, aminophylline, ethanol, propylene glycol,
`sodium pentobarbital, Tween 80.
`Group 3.-Suhstances whose isotonic concentra-
`tion is higher than their iso-osmotic concentration :
`dextrose (as regards human red cells), glycine,
`sodium salicylate.
`Group 4.-Substances which increase the per-
`meability of the erythrocyte membrane to NaCl:
`procaine hydrochloride, adiphenine hydrochloride,
`ethanol, and propylene glycol a t higher than 10-
`20y0 concentrations, Tween 60.
`Group 5.-Substances with a pronounced he-
`molytic action : saponin, sulfuric esters of methyl-
`androstenediol and of testosterone.
`Group 6.-Substances which exert a protective
`action similar to that exerted by dextrose as regards
`the increase in permeability caused by procaine :
`dextrose, sorbitol.
`precipitating proteins:
`Group 7.-Substances
`ZnSOd and all precipitants of proteins.
`
`render the solution isotonic it is not sufficient to add
`0.9% of NaCl but it is necessary to add this salt at a
`concentration of about 1.376, as if the procaine in-
`creased the permeability of the membrane to sodium
`chloride.
`It is interesting to note, however, that it is
`enough to add a 3.3% solution of dextrose (0.6 iso-
`osmolar) to the same solution of procaine hydrochlo-
`ride to have a solution which is isotonic for rabbit
`red cells, as if dextrose not only abolished the per-
`meabilizing effect of procaine but rendered the cell
`membrane partially impermeable to procaine.
`
`substance has an intense hemo-
`Saponin.-This
`lytic effect up to a concentration of 0.005-0.001~0
`even if dissolved in an 0.9y0 solution of NaCI. The
`behavior of rabbit red cells placed in NaCl and dex-
`trose solutions a t different concentrations and in the
`presence of saponin a t 0.059; was checked, and the
`results were very similar to those obtained with pro-
`caine hydrochloride at 5.05%.
`ZnSOa.-Zinc
`salts are of particular interest inas-
`much as Hartman and Husa (6) observed by means
`of their hemolytic method that ZnSOa “protects” the
`red cells to such an extent that the isotonic solution
`of the salt would be 400 times more dilute than the
`iso-osmotic concentration. Cadwallader and Husa
`(12) have described similar results for zinc acetate.
`By our method, however, it can be shown that ZnSO,
`precipitates plasma proteins and causes hemolysis up
`to a concentration of 155 mM. The results de-
`scribed by Hartman and Husa can be confirmed only
`if blood and ZnSO4 solution are mixed in the volu-
`metric proportion of 1 :50 (as these authors did), but
`it can also be demonstrated that the absence of lak-
`ing is due to a precipitation and a denaturation of
`hemoglobin. One cannot, therefore, accept the con-
`clusion that solutions of ZnSO4 are isotonic a t con-
`centrations 400 times lower than the iso-osmotic con-
`centration because not a protective action but a pre-
`cipitating and denaturating action by the zinc ion is
`implicated.
`Effects of the pH of Solutions on Intracellular Os-
`motic Pressure.-At
`physiological pH values nega-
`tive charges prevail in red cell hemoglobin and about
`50 meq./L. of cations are required for electrical neu-
`tralization. As hemoglobin is an ampholyte, its
`negative charges diminish when the environment be-
`comes acid, releasing cations which, being unable to
`diffuse in the extracellular fluid through the cell
`membrane which is impermeable to them, attract
`anions from the extracellular fluid. A diminution
`of pH, therefore, involves a rise in intracellular
`osmotic pressure and, conversely, an augmentation
`of pH causes a fall in intracellular osmotic pres-
`sure. The isotonic concentration must, therefore,
`depend to some extent upon the pH of the solution.
`Figure 3 gives experimental proof of this hypo-
`thesis. Although the cations concentration of the
`solution under examination was kept constant
`(155 meq./L. of Na+), it may be observed that the
`cell volume increases in acid solutions and decreases
`in alkaline solutions, demonstrating that the osmotic
`pressure of red cells increases in contact with acid
`solutions and decreases in contact with alkaline
`solutions. Here again the osmotic concentration of
`the various solutions, measured by determining the
`depression of their freezing points, was not closely
`related to the osmotic pressure of the solutions.
`
`SUMMARY
`
`When wishing to render a n injectable solution
`isotonic, the main consideration should be the
`permeability of the cell membrane to the various
`solutes composing the solutions and the action of
`these solutes upon the cell membrane. In other
`words, iso-osmia (which can be determined by
`physical methods based on one of the colligative
`properties) is equal to isotoniu only when all the
`solutes of the solution are unable to diffuse freely
`If this is not the
`through the cell membrane.
`case, isotonia can be determined only by measur-
`ing the osmotic effect of a given solution directly
`upon the concerned cells.
`
`~~
`
`REFERENCES
`(1) Meschia. G . , and Setnikar, I., J . Gen. Physiol., 42,
`429(1958).
`( 2 ) Szekely, I. J., and Goyan, F. M., THIS JOURNAL, 41,
`miwm
`(3) Husa W. J.. and Adams J . R. ibid. 33 329(1944).
`(4) GrosiLki, T . S., and Husa, W. i., i b i i . , 45, ti32(1954).
`( 5 ) Easterly, W. D., and Husa, W. J., ibid., 43, 750
`I 1 4.54) ~ _ - - _, .
`(0) Hartman, C. W., and Husa, W. J.. ibid., 46,430(1957).
`(7) Thomasson, C. L., and Husa, W. J . , ibid.. 47, 711
`(1958).
`(8) Cadwallader, D. IS., Jr., and Husa, W. J . . ibid.. 47,
`705(1958).
`(9) Wakes. F.. Qnarl. J . Pharm. and Pharinacol., 9, 455
`(1930).
`(10) Eijkman, C., Virchow’s Arch. palhol. Anal. u. Physiol.,
`143, 448(1896).
`(11) Ebina, R., Keijo J . M e d . , 8, 360(1937).
`(12) Cadwallader, D. E., Jr., and Husa, W. J., THIS
`JOURNAL, 47, 703(1988).
`
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