Effect of the venom of the bee Apis mellifera on osmotic fragility of cattle RBC

Toxtcon, 1968, Vol. S. pp. I81-186. Peraamon Pana Ltd., Printed in Chut Hrltain

EFFECT OF THE VENOM OF THE BEE APIS MELLIFERA ON OSMOTIC FRAGILITY OF CATTLE RBC I. HoRT and A. Hmtz Department of Physiology and Anatomy, The Hebrew University Faculty of Agriculture, Rehovot, Israel (Acceptedjor publication 11u1y 196 Abstract-The effect of bee venom on the osmotic fragility of cattle RBC was established using a method of recording the gradual osmotic hemolysis of the erythrocytes . The relative values of the RBC populations were obtained by direct planimetration of the area under derivative curves . Increased fragility and fusion of the two RBC populations into one were determined in relation to the venom concentration and the time interval during which the venom was present in the RBC suspensiôn . The fragiligrams were typical and reproducible, suggesting a new test for the effects of bee venom on blood. INTRODUCTION

er~ecT of bee venom on the osmotic fragility of human and rabbit RBC has recently been studied, using a fragiligraph [1-5]. The fragiligram was found to be a function of the salt concentration in the medium surrounding the erythrocytes, the time thevenomwas present in the RBC suspension, and the concentration of the venom in the suspension . Bee venom was found to increase the osmotic fragility and induce the division of the `adult' RBC into two or three populations having different osmotic fragilities- Changes in the relative values of the populations were related to the bee venom concentrations . In the present study the effect of bee venom on the osmotic fragility of cattle RBC was determined . Ti->B

MATERIALS AND METHODS

Six adult cows and 6 calves of 3-5 months of age were studied. Blood was drawn by vein puncture from the external jugular. Normal fragiligrams were obtained by a method based on gradual hemolysis in hypotonic NaCI solutions [1-3]. The blood was collected in heparinized capillary tubes, 7~5 mm long and 1~2 to 1~4 mm in dia., of the type used for the microhematocrit. A vol. of0075 ml of 1 :10 dilution of whole blood in isotonic, buffered NaCI solution was introduced into a container cell, the walls of which were made of a dialyzing membrane. The container cell was introduced into a test tube of distilled water placed in a colorimeter with a recorder, between a source of light and a photoelectric cell. Dialysis through the membrane resulted in a continuous decrease in the salt concentration of the medium surrounding the erythrocytes. The measurement of degree of hemolysis was based on the increasing transparency of the erythrocyte suspension while hemolysis took place. Recording this increasing light transmission as a function of time, i .e. as a function of decreasing salt concentration, 181

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I. HORT and A. HERZ

yielded the fragiligram or its derivative . The salt concentration corresponding to each point on the cumulative curve was found with the aid of an established curve [2]. The effect of the venom was studied by mixing 1 ml of a buffered isotonic NaCI solution containing 20 y per ml of bee venom with different volumes ranging from 1 to 999 ml of 1 :10 RBC suspensions in buffered isotonic NaCI solutions. After 20-30 sec, 0075 ml of the suspension was introduced into the fragiligraph for recording. A few minutes later, a second and sometimes a third record from the sample was made (tests 1, 2 and 3). The relative values of the RBC populations were obtained by direct planimetration of the area under derivative curves. RESULTS

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Bee Venom

183

was present in the RBC suspensions. The time values were transferred to concentration values. The fragiligrams and the fragility values for adult cows are presented in Fig. 1 and Table 1 . TABLE

Concentration Controls lilt 1 :2 1 :5 1 :10 1 :100 1 :1000

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Test No. 1 1 2 1 2 1 2 3 1 2 3 1 2 3 1 2 3

Time interval'

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time (min)

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0"30f0 "09 0"30f0 "03 4"30f0~70 0"30f0 "09 6"OOfI "35 0"30f0 "04 6"30~0 "S1 23"OOf4 "S1 0"30 f0"03 7"200"07 16"OOf2 "50 0"300"03 11 "001 "53 18 "30f2"S1 13 "00ß"S8 23 "0Oß"88 32"OO~S "30

1"4f0"20 0"Of0"00 0"Of0"00 0"Of0"00 0"00 "00 1"Of0"03 0"40 "02 0"00 "00 l "00 "23 0"70 "07 0"7f0"12 1"2f0"03 1"Of0"09 0"7f0"08 l"6f0"24 1"S~0"09 1"2f0"10

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3"80"28 0"S~0"03 0"80"10 2"6~0"S1 . 1"910"27 1"20 "10 3"2f0 "2S 2"710"78 2"40 "23 3"7f0"37 3"00 "40 2"7f0"30 4"Sf0"SO 3"S~0"72 3"S~0"S1

0"36 0"78 0"4S 0"S3 0"66 0"40 0"44 0"47 0"37 0"42 0"44 0"37 038 0"38

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The time (concentration~hemolysis curves of the controls had a sigmoidal pattern, and the derivative curves were unimodal, as typical of a continuous distribution of a heterogeneous population . The fragility values were within the normal range [6]. The cumulative and derivative curves for the venom studies were similar in pattern to those of the controls . The fragility values showed increased fragility of the RBC. The fragility of the erythrocytes was increased as the time interval from the mixing to the test point was extended (from test 1 to test 3). The cow RBC were not divided into two populations, as was found for human and rabbit erythrocytes [4, S]. In the calves, the fragiligrams and their fragility values are summarized in Fig. 2 and Table 2. The relative values of the RBC populations are presented in Table 3. In the controls the cumulative and derivative curves were bimodal as typical for normal calves [7]. The fragility values were within the normal range. In the venom studies an increase in the fragility of the RBC population, fusion of the two populations into one and changes in the relative values of the populations, were found according to the different venom concentrations and tests. The fragility of RBC was increased from the lowest to the highest venom concentrations and from the first to the third tests .

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TABLE 3. THE RELA7IVS VALUES OF I'HE

Concentration Controls 1 :50 t 1 :100

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RBC i'OYULATIUNS (peC ~~nt) UHTAINED FROM AND THE CONTROLS (CALVES)

Time interval " (min)

Population (/)

0"30--x-0"09 0"500" 04 30~00.~,,- 5"80 60 "OOt9 "53 0"40f0 "09 11 "OOf0~90 54f00s3~35

4323 " 3 542f6"1 50"9-5"8 50"09 "0 53 "52"5 49 "3-±-3"4 54"84 "6

THE TREATMENT STUDIES

Population (gô) Sb"9fb"3 458-!-3"7 49"1 -~ 8" U 500.:=6"1 46 "5 :6"3 50 "7-~-9"9 452-!:4" 8

Fragiligram form 'Time the venom was present in the RBC suspension . tVol . of venom suspension/total vol. of blood and venom suspensions . DISCUSSION

In general, the same results seen in the human and rabbit studies are demonstrated here for cattle RBC. The fragiligrams were found to be a function of the NaCI and bee venom concentrations in the suspension surrounding the erythrocytes, and the time interval during which the venom was present in the suspension . However, division of the `adult' type RBC was not found. Similar to the findings for human and rabbit RBC, the fragiligrams of cattle RBC in the presence of bee venom were typical and reproducible and suggest the value of the test for the effect of the venom on blood. [1] [2] [3] [4] [5] [6] [7]

REFERENCES A, and MARIKOVSKY, Y., Preparation of erythrocyte ghosts by gradual hemolysis in hypotonic aqueous solution . Bull. Res. Couac. Israel. 6E, 36, 1956. DANON, D., A rapid micro method for recording red cell osmotic fragility by continuous decrease of salt concentration. J. clin. Path. 16, 377, 1963 . DANON, D., FRET, E. H., FRET, Y. F. and LIPKIId, Y., An instrument for automatically recording the osmotic fragility Curve of red blood cell, and/or its derivative. 7ians. IEEE Blol. Med. Elect., BME-10 . 1, 24, 1963. HoRT, I. and HERZ, A., The influence of bee venom on the osmotic fragility of human RBC. F.xperientia. In press, 1968. HoRT, I. and HERZ, A., The influence of bee venom on the osmotic fragility of rabbit RBC. Experientia . Tn press, 1968 . PERK, K., FRET, Y. F. and HERZ, A., Osmotic fragility of red blood cells of young and mature domestic and laboratory animals. Am. J. vet. Res. 2S, 1241, 1964 . FhEr, Y. F., Pte, K. and DANON, D., Correlation between osmotic resistance and fetal hemoglobin in bovine erythrocytes . J. exp. Cell Res. 30, 561, 1963 . DANON, D., NEVO,