The Effect of Zinc Acetate on Red Corpuscles*

The Effect of Zinc Acetate on Red Corpuscles* By DONALD E. CADWALLADER, Jr.,t and WILLIAM J. HUSA In hemolysis experiments, unusual behavior was displayed by solutions of zinc acetate. Hemolysis of red blood cells took place at an unusually high concentration range while extremely low concentrations of zinc acetate protected red blood corpuscles against osmotic hemolysis. The results are graphically portrayed.

HUSA(1) found extremely high hemolytic i values for zinc sulfate. They were of t h e opinion that zinc sulfate strengthens the erythrocyte membrane in some way, making the red cell more resistant t o hemolysis i n hypotonic solution. T h e purpose of this investigation was to observe the effect of a zinc salt of an organic acid on rabbit a n d human red hlood cells. ARTMAN AND

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EXPERIMENTAL Collection of Blood, Preparation of Solutions and Quantitative Determination of Per Cent Hemolysis. -The procedures employed were essentially those described by previous workers (2, 3). Preparation of Graphs.-Three sodium cliloridc experiments employing rabbit blood were carried out. The average readings of these three experiments were utilized to construct one standard sodium chloride curve, Fig. 1. The average readings of three zinc acetate experiments, using the same rabbit blood samples as the above sodium chloride experiments, were employed to construct Fig. 2. Therefore, Fig. 1 is the standard sodium chloride curve for Fig. 2, the zinc acetate curve. Experiments were run with rabbit blood employing sodium chloride solutions and comparatively low concentrations of zinc acetate. A bell-shaped hemolysis curve was not formed with the lower concentrations of zinc acetate as was the case when higher concentrations of zinc acetate were used, Fig. 2. A dilution range of zinc acetate was employed so that approximately 5 to 100% hemolysis was obtained. Using Eq. 1, hemolytic i values were calculated from these zinc acetate experiments. These hemolytic i values are shown in Table I.

.48

a

Blood

35

50

Rabbit Human

718 58.3

835 568

7a--

.46

.45 .44 . 4 3 .42 , 4 1 . 4 0 .39 PER CENT NaCl

.38

Fig. 1.-Standard sodium chloride curve for hemolysis of rabbit erythrocytes.

TABLEI.-VALUES FOR i FOR ZINC ACETATE AT CONCENTRATIONS, IN G ~ . / 1 0 0ML., CAUSING25, OF RABBIT AND HUMAN 50, A N D 75y0 HEMOLYSIS ERYTHROCYTES" --Hemolysis,

.47

0

1.70 1.60 1.50 1.40 1.30 1.20 1.10 1.00 .90 .80

.70

75

Average

PER CENT ZINC ACETATE

902 648

818

Fig. 2.-Hemolysis of rabbit erythrocytes in zinc acetate solutions of varying concentrations.

.. .

All i values represent an average of two blood samples.

* Received January 4, 1958, from the College of Pharmacy, University of Florida, Gainesville. This paper is based in part upon a dissertation presented t o the Graduate Council of the University of Florida by Donald E. Cadwallader. Jr., in partial fulfillment of the requirements for the degree of Doctor of Philosophy. t Fellow of the American Foundation for Pharmaceutical Education 1954-1957. Research Associate, Sterling-Winthrop Research Institute, Rensselaer, N . Y. Presented to the Pharmacy Section, A. A. A. S., Indianapolis meeting, December, 1957.

The construction of Fig. 3 consisted of two parts. The left hand portion of the curve is Fig. 2 transposed to the log scale. In order to make up the right hand portion of the curve, the Gm./100 ml. of zinc acetate causing 25, 50, and 75% hemolysis, at comparatively low zinc acetate concentrations, had to be calculated with reference to Fig. 1. Utilizing the following equation 703

704

AMERICANPHARMACEUTICAL ASSOCIATION Vol. XLVII, No. 10

JOURNAL OF THE

.

.

in 100 ml. o f solution

701

-

(Gm.-molecular weight of iYaC1)

(

i value for

)(

F5

Gm. of zinc acetate in 100 ml. of solution

)

zinc acetate (Gm.-molecular weight of Zn(A4c)t) ~

0%. 1)

the Gm./100 ml. of zinc acetate causing 25 per cent hemolysis was calculated by substituting 1.86 for the i value of sodium chloride, Gm./100 ml. of sodium chloride, determined from Fig. 1, causing 257, hemolysis, and the hemolytic i value for zinc acetate for 25% hemolysis (see Table I), and then solving for Gin. of zinc acetate in 100 ml. of solution causing hemolysis. Similarly the C:m./100 ml. of zinc acetate, causing 50 and 75% hemolysis, were calculated by substituting the appropriate values in Eq. 1. The above three points were used to construct the right hand portion of Fig. 3. I n this manner, Fig. 3 becomes a compilation of all the zinc acetate experiments run with rabbit blood, and Fig. 1 is the standard sodium chloride curve for Fig. 3 . The same procedure was carried out for human blood using Fig. 4 as the standard sodium chloride curve. Figures 4, 5, and 6 were constructed from the average of four experimental runs. -0.3 -0.2

DISCUSSION In the hemolysis experiments, unusual behavior was displayed by solutions of zinc acetate. Figure 2 shows the hemolysis curve when rabbit erythrocytes were placed in various concentrations of zinc acetate. Starting in 1.7% zinc acetate solution, hemolysis increased to a maximum of 42% in 1.37% zinc acetate solution and then decreased until only 596 hemolysis was observed in 0.77, solution. The same behavior was noticed for human blood and is plotted in Fig. 5. However, the maximum hemolysis was 95% in l.lOyG zinc acetate solution. Hemolysis of human blood took place over a lower concentration range than rabbit blood; 1.60 to 0.40% zinc acetate solution. Zinc acetate solutions ranging from 0.70 to 0.017& displayed essentially complete protection against hemolysis of rabbit erythrocytes. Human erythrocytes were protected in solutions ranging from 0.40 to 0.1%. Upon further lowering o f the zinc acetate concentration, hemolysis increased rapidly in the case of rabbit blood. Fur human blood 250/, hemolysis occurred in approximately 0.05% solution, then there was a leveling off with hemolysis commencing to increase rapidly a t a concentration of 0.01%. Extremely high i values were found for zinc acetate using data obtained from the heniolysis experiments. Higher hemolytic i values were found for rabbit blood. The p H of zinc acetate solutions used in hemolysis experiments was slightly acid. No unusual deviations of pH were observed for the various solutions. In 1931 Jaeger (4)reported that the heniolysis of horse erythrocytes by saponins or water was inhibited by zinc acetate. He also stated that chicken protein and horse serum were precipitated and that

-0.1 0.0 0.1 0.2 2.3 -log c C = Gm. zinc acetate/100 ml.

2.4

2.5

2.6

Fig. 3.-Hemolysis of rabbit erythrocytes in zinc acetate solutions of varying concentration.

I

62

!!

t

/

70C

.44

.43

.42 . 4 1 ,40 . 3 9 .38 PER C E N T NaCl

37 , 3 0 .35 . 3 4

Fig. 4.--Standard sodium chloride curve for hernolysis of human erythrocytes. lipoids (cholesterol and lecithin) were almost quantitatively precipitated by zinc ion. Hartman and Husa (1) found unusually high i values for zinc sulfate. Red cells take up zinc very readily (5). Whether the zinc enters the red cell or is bound to the membrane is not known, but it cannot be removed by washing with isotonic saline. Gurd and Wilcox (6)

October 1958

SCIENTIFIC Enr-r~os

2

5 0

ion 90

so 70 GO

x

50

ff

40

&

30 20

: 2

10

0 -0.2

l0 o

LI

1.50 1.40 1.30 1.20 1.10 1.00 .90 .80 .70 .GO .50 .40 P E R C E N T ZINC ACETATE

Fig. 5.-Hemolysis of human erythrocytes in zinc acetate solutions of varying concentrations.

list the postulates which lie behind the computations for the binding theory of zinc-albumin systems at 0’. The postulates are: (a) Zinc ion is bound to only one class of sites within the experimental range of pH. (b) The interdependence of zinc ion binding and pH reflects a competition between zinc ion and hydrogen ion for the same ligand groups. (c) The ligand groups in question are the imidazole side chains of the histidine residues. (4 Complex formation between zinc ion and each of the imidazole groups of the albumin molecule occurs in the ratio of 1:l.

0.2 0.6 1.0 1.4 1.8 2.2 2.6 8.0 0.0 0.4 0.8 1.2 i . ~ 2.0 2.4 2.8 -log C C = Gm. zinc acetate/100 ml.

Fig. 6.-Hemolysis o f human erythrocytes in zinc acetate solutions of varying concentrations. SUMMARY 1. In hemolysis experiments with rabbit and human blood, unusual behavior was observed for zinc acetate. 2. Extremely high i values were calculated for zinc acetate using hemolytic data.

REFERENCES !l> Hartman, C . W., and Husa, W. J., THIS JOURNAL, 46, 430(1957). (2) Grosicki, T. S., a n d Husa, W. J., ibid., 43, 632(1954). (3) Easterly, W. D. a n d Husa W. J. ibid. 43 753(1954). (4) Jaeger. H.. AT&. expll. Pn‘fhol. Pharmbkoi., 159, 139 c ioqi) \.“v.,. (5) Tullis, J . I-., “Blood Cells and Plasma Proteins-Their State in Nature.” Academic Press Inc.. New York. 1953.. D. _ 202. (ti) Anson, M. I,., Btily, K., a n d Edsall, J. T., “Advances in Protein Chemistry, Volume X I . Academic Press Inc., New York, 1956, pp. 376-377.

Isotonic Solutions VI. The Permeability of Red Corpuscles to Various Salts of Organic-Acids* By DONALD E. CADWALLADER, Jr.,t and WILLIAM J. HUSA Employing the hemolytic method, van’t Hoff i values were obtained for various salts of organic acids. Results indicate that sodium and potassium salts of citric, tartaric, and succinic acids have higher hemolytic i values than might be expected. Ammonium citrate and tartrate have higher i values than the corresponding sodium and potassium salts. Ammonium acetate, benzoate, and salicylate, and bismuthpotassium tartrate caused 100 per cent hemolysis in all concentrations tested. This illustrates the need for caution in the use of isotonic coefficients obtained from colligative properties alone. HE PREPARATION of solutions isotonic with Thuman blood is of prime importance to the safety and comfort of the patient. Much information has been compiled concerning the

*

Received January 4.1958, from t h e College of Pharmacy, University of Florida, Gainesville. Presented t o the Pharmacy Section, A. A. A. S., Indianapolis meeting, December, 1957. This paper is based in p a r t upon a dissertation presented t o t h e Graduate Council of the University of Florida b y Donald E. Cadwallader, Jr.. in partial fulfillment of the requirements for the degree of Doctor of Philosophy. t Fellow of the American Foundation for Pharmaceutical Education 1954-1957. Research Associate, Sterling-Winthrop Research Institute, Rensselaer, N. Y.

use of colligative properties of drugs in the calculation of isotonic solutions. Working with isotonic solutions, Husa and Adams (1) pointed out that certain substances a t concentrations calculated to be isotonic with blood, according to physicochemical data, did not prevent hemolysis. A hemolytic method for the quantitative determination of sodium chloride equivalents was employed by Husa and Grosicki (a),and isotonic coefficients or i values of various amino acids, sugars, and salts