The osmotic fragility of some fish erythrocytes in hypotonic saline

Camp. Biochem. Physiol., 1969, Vol. 28, pp. 409 to 415. Pergamon Press. Printed in Great Britain

THE OSMOTIC F R A G I L I T Y OF SOME FISH ERYTHROCYTES IN H Y P O T O N I C SALINE* GEORGE H. EZELL, L. L. SULYA and C. L. D O D G E N Department of Biochemistry, Universi~" of Mississippi Medical Center, Jackson, Mississippi (Received 9 Nlay 1968)

Abstract--1. Osmotic fragility curves of erythrocytes from eleven species of marine teleosts from the Gulf of Mexico are of the normal sigmoid type. 2. The mean corpuscular fragility value is generally consistent within a species but varies widely among the different species. 3. Two species of gars, Lepidosteus osseus and L. productus, have erythrocytes which are resistant to hemolysis in hypotonic sodium chloride solutions. INTRODUCTION ALTHOUGHthere is considerable literature concerning the biochemisty of hemolysis, especially from the standpoint of hemolytic disease (Carson & Tarlov, 1962), investigations of the hemolysis of nucleated red cells are few (Kariya, 1950; Sulya et al., 1961, 1963 ; Frei & Perk, 1964). In investigating the osmotic hemolysis of the carp, Cyprinus carpio, Kariya (1950) observed a hemolygram similar to that of mammals. Frei & Perk (1964) studied the osmotic hemolysis of erythrocytes from chickens and found two different types of osmotic fragility curves for laying hens and mature cocks. In each case the minimal osmotic resistance occurred at about 0.340/0 NaCI with maximum hemolysis at 0.25% NaC1. However, the shapes of the fragility curves were not the same after maximum hemolysis had occurred. Hemolysis remained maximal with the erythrocytes of the mature cocks, whereas hemolysis appeared less than maximal at NaC1 concentrations less than 0.25 per cent for the erythrocytes of the laying hens. They presented evidence for the existence of a factor in the plasma of the laying hens which caused aggregation of the hemolyzed cells resulting in an increased turbidiw of the solution. By their method of measuring hemolysis, such an increase in turbidity would result-in an "apparent" decrease in erythrocyte fragility. Sulya et al. (1961), in collecting blood from marine fishes, observed hemolysis under conditions which would be expected to yield a hemoglobin-free plasma or serum with mammalian blood. This observation, that the erythrocytes of marine fish were apparently more fragile than mammalian erythrocytes, led them to make a preliminary investigation of the hypotonic fragility of a few species of teleosts * This investigation was supported by Public Health Service Research Grant HE 0989802 from the National Heart Institute. 409

410

GEORGE H. EZELL, L. L. SL'LYAAND C. L. DODGF-N

f r o m the G u l f of Mexico (Sulya et al., 1963). T h e i r results indicated that the hemolytic fragility curves of these fish were generally quite different f r o m those o b s e n ' e d in m a m m a l s and suggested a n o n u n i f o r m population of erythrocytes in several species. I n addition, their resuks suggested that the hemolysis of the erythrocytes in distilled water was incomplete. T h e s e preliminary findings suggested the need for a m o r e detailed investigation of this apparent unusual e r y t h r o c ~ e fragility in marine fish. T h e results of such an investigation regarding the m e a s u r e m e n t of the osmotic fragility of fish erythrocytes in h y p o t o n i c saline solutions u n d e r carefully controlled experimental conditions and the m e a s u r e m e n t of other parameters, such as the erythrocyte c o u n t and hematocrit, are presented in this paper.

MATERIALS AND METHODS Experimental animals Marine fish representing six families and eleven species were caught in the Mississippi Sound of the Gulf of Mexico near Ocean Springs, Mississippi. The specimens were selected to represent species of differing evolutionary development and er?-throcyte fragility patterns.

Collection of blood Immediately after capture, the caudal peduncle was rinsed with distilled water and blotted dry. The caudal artery was severed and the blood collected in glass vials containing two drops of dipotassium ethylene diaminotetracetate as an anticoagulant. The blood was then kept in an ice bath until all the analyses had been completed. The tests were begun as soon as possible after the collection of the blood, 4 hr being the maximum holding time. ,,lleasurement of osmotic fragility The method used was a modification of the procedure of Parpart et al. (1947). A stock solution of sodium chloride in phosphate buffer was prepared which was equivalent to a 10% solution of NaC1. The pH of this solution was 7"40. Working solutions were prepared from this stock solution by diluting with distilled water so that a range of concentrations from 0"00% to 1"00%, in increments of 0'05% NaC1, were used. The test was performed by adding 0"025 ml of blood to 5'0 ml of each of the working solutions. The blood was mixed with the salt solutions by gently inverting each tube three times. Each solution was then allowed to incubate at 26 _+I°C for 40 rain. At the end of the incubation period, the solutions were centrifuged for 10 rain in an Adams Safeguard Angle-Head Centrifuge, model CT-1230 with CT-1235 head, at 2200 rev/min. The percentage hemolysis was determined by measuring the optical density of the supernatant hemoglobin solution at 540 m/~ in a Bausch and Lomb Spectronic 20 colorimeter with maximum hemolysis corresponding to the solution having the highest optical density reading. If the incubation is carried out in calibrated spectrophotometer tubes, it is not necessary to remove the sedimented material before reading the optical density. Determination of microhematocrit The packed red blood cell volumes of the anticoagulated blood was measured in microhematocrit tubes by centrifuging the blood at 2200 rev/min for 30 rain. The results are expressed in terms of the percentage of total blood volume.

OSMOTIC

FRAGILITYOF FISHERYTHROCYTES

411

Blood cell counts The blood was diluted with Shaw's solution to stain both the erythrocytes and leucocytes, and counting of the cells was done using a Spencer Brite-Line hemac.vtometerof the improved Neubauer type. The results are expressed as the number of red and white blood cells per mm3. RESULTS AND DISCUSSION In Fig. 1, examples of the results are expressed in the conventional way by plotting the percentage hemolysis against the concentration of sodium chloride.

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z

60

20 30 40 50 60 70 SODIUM CHLORIDE CONCENTRATION PER CENT X 10 2

80

90

FIG. 1. Osmotic fragili~- curves of fish red cells. A, Lepidosteus productus (spotted gar); B, Paralichthys lethostigmus (flounder); C, Galeichthys fel# (sea catfish); D, Micropogan undulatus (croaker). These graphs show clearly that the shape of the hemolysis curves for these fish is of the sigmoid type obtained with human blood. This is in contrast with the previous findings (Sulya et al., 1963) which indicated that the only species which approached the sigmoid type of curve were the gars. However, the sodium chloride solutions used in the preliminary experiments were not buffered as were the sodium chloride solutions used in the present experiments. Since Parpart et al. (1947) have shown that the hydrogen ion concentration of the solution markedly affects the degree of hemolysis, it is possible that variability in the pH of the test solutions was responsible for the deviation from the normal sigmoid type of curve during the preliminary experiments. In addition, we have observed in the present experiments what we propose is a mechanical fragility of the erythrocytes of several species, i.e. Micropogan undulatus, Menticirrhus litteralis, Mugil cephalus and Archosargus probatocephalus. In most of the specimens from these species, the osmotic fragility curves obtained were of the sigmoid type, but in several of the specimens a radical departure from the sigmoid curve was observed at high concentrations of NaC1. The type of curve obtained is shown in Fig. 2. Even with a nonuniform cell population, the

GEORGEH. EZELL,L. L. SULYAA N D C. L. DODGEN

412

degree of hemolysis must be greater at a lower NaC1 concentration than at a higher NaC1 concentration for the same sample of blood. That this is not the case for these specimens is evident from Fig. 2. Obviously then, another variable other than changing NaC1 concentration, has been introduced. It is our opinion that this artifact may be caused by erythrocytes which possess an intrinsic mechanical fragility, and that the artifact will not appear if the erythrocytes are handled with

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Anomalous osmotic fragility curve of ArchosaGus probatocephelus (sheepshead). Dashed line represents normal sigmoid curve.

extreme care during the collecting and mixing procedures. Further, it seems likely that such a mechanical fragility might be due to a difference in the celt membrane structure and/or composition in these species. Further evidence for this increased susceptibility towards hemolysis of the erythrocytes of these species comes from the fact that out of eighty-four specimens from these three species, twenty-one specimens showed maximal or nearly maximal hemolysis even at a NaC1 concentration of 1.00% (Table 1). In Fig. 3 the data from Fig. 1 have been plotted in the form of hemolytic increment curves (Suess et al., 1948). Monophasic increment curves are obtained on normal mammalian blood, but biphasic curves are found in cases of spherocytosis, and are believed to be due to a nonuniform cell population. The graphs in Fig. 3 are of the monophasic type and indicate a uniform cell population for these species of marine fish. Again, this is in contrast to the hemolytic increment curves obtained in the preliminary studies (Sulya et al., 1963). However, since the increment curve is the derivative form of the sigmoid curve (percentage hemolysis vs. NaC1 concentration), a curve which was not sigrnoid would not yield a monophasic increment curve. It has already been mentioned that these curves were not sigmoid in the preliminary studies, due possibly to variations in the pH of the test solutions or to mechanical fragility of the erythrocytes. Further examination of the curves in Fig. 1 reveals that the osmotic fragility patterns of the blood from different species are not the same. Some species have

OS.MOTIC

TABLE 1--.'%lEAN C O R P U S C U L - k R OF

FRAGILITY FR.-~GILITY~

TE2q S P E C I E S

No. of fish*

Family and species

OF FISH

ERYTHROCYTE OF

413

ERYTEROCYTES

MARLNE

COI.,~'~'TS .A.ND M I C R O H E . % L ~ T O C R I T S

TELEOSTS

MCF (~ + S.D.)

% HET (~ + S.D.)

Erytahrocytes x 10-4/ram 3 (~ + S.D.)

Lepidosteidae

Lepidosteus osseus Lepidosteus productus

2 1

0-40 0"34

40 33

134 120

21

0"57+0"05

32+4

109_+18

23 s

0'68 + 0"13

33 + 4

271 _+38

6 40 ~ 83 4

0'65 _+0"05 0"61 +0-13 0"56 -+0"04 0"53 + 0'02

36 _+3 32_+4 34 + 4 34 -+ 6

259 _+39 257+25 256 -+46 314 -+91

133

0"64 + 0'08

38 -+ 3

268 _+69

15

0"53 _+0"04

27 _+3

215 + 42

Ariidae

Galeichthysfelis Mugilidae

i]/lugil cephalus Sciaenidae

Sciaenops ocellata 3¢icropogan undulatus :'Vlenticirrhus litteralis Cynoscion arenarius Sparidae

Archosargus probatocephalus Bothidae

Paralichthys lethostigmus

* Superscripts to number of fish represent the number in this group having M C F value greater than 1"00% NaCI and thus were omitted in determining mean M C F values.

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FIG. 3. Hemolytic increment curves. Increment of hemolysis is change in percentage hemolysis per 0"05% change in sodium chloride concentration. A, Lepidosteus productus (spotted gar); B, Paralichthys lethostigmus (flounder); C, Galeichthys felis (sea catfish); D, Nlicropogan undulatus (croaker).

414

GEORGEH. I~ZELL,L. L. S<-LY.{.~.\'D C. L. DODGEN

erythrocytes that are quite resistant to hypotonic hemolysis, while others have a much more fragile erythrocyte population. The Mean Corpuscular Fragility (MCF), which is the concentration of sodium chloride in which one-half of the erythrocytes are hemolyzed, may be used as an index of the osmotic fragility" of the erythrocytes. Table 1 lists the M C F of each species studied, along with the average red blood-cell count and the average microhematocrit of each species. The erythrocytes which exhibited the most resistance to hypotonic hemolysis were those of the spotted gar, Lepidosteusproductus, having a M C F of 0.34°.0 NaC1, Only one specimen was available from this species, but it compared well with two specimens from this same species in the preliminary experiments which had M C F values of 0.35 and 0.32°o NaC1. The individual M C F values of the two longnose gars, L. osseus, were 0.37 and 0.42°.0 which compared with three values from this species in the preliminau experiments of 0.40, 0.44 and 0.46°0 NaC1. Most of the specimens from the other species had M C F values ranging from 0"50°o NaC1 to 0.68°,i; NaC1. In addition, the M C F values for the same species generally agreed well with one another; i.e. 0.53°'o with a standard deviation of 0.04% for fifteen specimens of Paralichthys lethostigmus and 0.57°o with a standard deviation of 0.05oo for twenty-one specimens of Galeichthys fells. Ervthrocvtes having mean corpuscular fragilities greater than 0.70% NaC1 were encountered in several species as seen in Table 1. These were the same species mentioned previousIy as possessing erTthrocytes with an increased susceptibility to hemolysis due to mechanical fragility. There does not appear to be a correlation between the osmotic fragility and the hematocrit nor a direct correlation between the fragility and the red blood count. However, there does seem to be a trend towards increased fragility as the red cell count increases.

Hemolysis in distilled water It can be seen in Fig. 1 that 100 per cent hemolysis always occurs at some concentration of NaC1 and remains 100 per cent at all lower concentrations of salt. However, when the percentage of hemolysis in pure water is based on this optical density value, it is always less than 100 per cent. This means that the amount of hemoglobin in this supernatant solution is less than the amount of hemoglobin in the supernatant solutions which represent 100 per cent hemolysis in hypotonic saline. This phenomenon was consistently observed and reported in the preliminaryexperiments (Sulya et al., 1963). Three possible causes of this anomalously low fragility are (1) some red cells remain unhemolyzed in water and thus do not release their hemoglobin into the supernatant, (2) complete hemolysis of the cells does occur, but part of the released hemoglobin is a water-insoluble macromolecule which is precipitated during the centrifugation process, or (3) part of the released hemoglobin is adsorbed on the water-insoluble nucleic acids, which are also released during hemolysis and precipitated during centrifugation. The first of the three possibilities did not seem likely, and observations of the cent_~uged material under the microscope did not reveal any unhemolyzed ceils. In order to test the water-insolubility theories, experiments were conducted in which hemolysis in

O S M O T I C F t L I . G I L I T Y OF F I S H E R Y T H R O C Y T E S

415

distilled water was carried out in the usual way, i.e. 0.025 ml blood/5-00 ml water. Simultaneously tubes containing 0.025 ml blood/2.50 ml water were prepared. At the end of the incubation period, 2.50 ml of 2.00°/0 NaC1 was added to the latter tube to adjust the final NaC1 concentration of the solution to 1"00 per cent. After mLxing the contents by inverting the tube several times, the tubes were centrifuged and the optical density of the supernatant hemoglobin solutions was read. T h e results showed that the optical density of water-hemolyzed supernatants was increased to values corresponding to 90-95 per cent of the maximum optical densities in hypotonic saline. Again no unhemolyzed cells were seen under the microscope. T h u s it appears that the apparently low fragility in water is an artifact, caused by either precipitation of macromolecular hemoglobin or partial adsorption of the hemoglobin by water-insoluble cellular material. No evidence has been obtained, thus far, which would indicate an aggregating factor in the plasma of fish blood similar to that proposed by Frei & Perk (1964) to explain an analogous situation in the blood of laying hens. SUMMARY 1. The osmotic fragility of the erythrocytes from eleven species of marine teleosts from the Gulf of Mexico has been measured, and the shape of the fragility curves obtained is of the normal sigmoid type. 2. The mean corpuscular fragility value is generally consistent within a species, but varies widely among the different species. 3. Two species of gars, L. osseus and L. productus, both seem to have erythrocytes which are resistant to hemolysis in hypotonic sodium chloride solutions. 4. The erythrocytes of several species of fish seem to have an unusually high intrinsic susceptibility towards hemolysis caused by mechanical trauma. 5. The anomalously low fragility of fish erythrocytes in distilled water appears to be caused by an artifact and not by incomplete hemolysis of the erythrocytes. REFERENCES CARSONP. E. 3: TARLOVA. R. (1962) Biochemistry of hemolysis. Ann. Rev. ~Vled. 13, 105126. FREI Y. F. 3: PERK K. (1964) Osmotic hemolysis of nucleated erythrocytes. Expl. Cell Res. 35, 230-238. KARIYA T. (1950) The blood corpuscle resistance of fish. Bull. Japan Soc. Sci. Fisheries 16, 65-69. PARPARTA. K., LORENZP. B., PARPARTE. R., GREGGJ. R. 3: CHASEA. l~l. (1947) The osmotic resistance (fragility) of human red cells. J. Clin. Invest. 26, 636-640. Su-Ess J., LI~rENT.~NID., DAMESCHEKW. 3: COLLOFF3,1. J. (1948) A quantitative method for the determination and charting of the erythrocyte hypotonic fragility. Blood 3, 1290-1303. SL'LYAL. L., Box B. E. 3: GL.'NTERGORDON(1961) Plasma proteins in the blood of fishes from the Gulf of Mexico. Am.J. Physiol. 200, 152-154. SULYAL. L., DODGENC. L. 3: CHRISTMASJ. Y. (1963) The hypotonic fragility, of some fish erythrocytes. J. Miss. Acad. Sci. 9, 275-282.