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Lipid concentrations in erythrocyte membranes in normal, starved, dehyrated and rehydrated camels (Camelus dromedarius), and in normal sheep (Ovis aries) and goats (Capra hircus)
ARTICLE IN PRESS Journal of Arid Environments Journal of Arid Environments 59 (2004) 675–683 www.elsevier.com/locate/jnlabr/yjare
Lipid concentrations in erythrocyte membranes in normal, starved, dehyrated and rehydrated camels (Camelus dromedarius), and in normal sheep (Ovis aries) and goats (Capra hircus) A.A. Al-Qarawi, H.M. Mousa* Department of Veterinary Medicine, College of Agriculture and Veterinary Medicine, King Saud University, Gaseem Branch, P.O. Box 10158, Buraydah, Al-Gaseem 81999, Saudi Arabia Received 8 January 2003; received in revised form 14 January 2004; accepted 10 February 2004
Abstract The present work compared the effects of starvation (7 days) and dehydration (10 days) followed by rehydration, on erythrocyte membranes of the desert camel (Camelus dromedarius). Total lipids, cholesterol, total phospholipids, phospholipid classes, fatty acids of phospholipid classes and proteins in the erythrocyte membranes of normal camels, sheep (Ovis aries) and desert goats (Capra hircus) have also been investigated. The results suggested that the lipid profile in the erythrocytes of the normal camels was significantly different from those found in normal sheep and goats. Between the two later species no significant differences were observed. In the erythrocyte membranes of the camel two classes of phospholipids (phosphatidylcholine and sphingomyelin), cholesterol and proteins were significantly higher than in sheep and goats. These constituents were not altered significantly by dehydration or starvation in the camel. These results may help to explain the resistance of camel erythrocytes to haemolysis after rehydration, infection with blood parasites or when confronted with other harsh environmental conditions. r 2004 Elsevier Ltd. All rights reserved. Keywords: Camel; Dehydration; Erythrocytes; Sheep; Goats; Saudi Arabia
*Corresponding author. E-mail address: [email protected] (H.M. Mousa). 0140-1963/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.jaridenv.2004.02.004
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1. Introduction Desert camels (Camelus dromedarius) are endowed with certain anatomical, physiological, biochemical and pharmacological peculiarities, which make them distinct from other related ruminants. For example, they are well known for their exceptional ability to withstand long periods of dehydration (MacFarlane et al., 1963; Perk, 1963), their ability to replenish lost water after long periods of dehydration in a relatively short period of time (10 l/min) (Schmidt-Nielsen et al., 1956), and to utilize low-quality roughages (Mousa et al., 1983). It is established that camel erythrocytes are resistant to haemolysis and can expand to about twice its original size when placed under hypotonic solutions (Perk et al., 1964). These properties were partly explained on the basis of their shape as camel erythrocytes are different from mammalian types in being oval, rather than circular discs (Turner et al., 1958; Jain and Keeton, 1974), and partly on their membranes (Livne and Kuiper, 1973; Mirgani, 1992). Several studies have attempted to study the ability of the camel to withstand dehydration and rapid rehydration (e.g. Ali et al., 1982; Mousa et al., 1983), and the contribution of the erythrocytes and their membranes composition to the physiological adaptation to dehydration (Livne and Kuiper, 1973; Eitan et al., 1976; Smith et al., 1979; Omorphos et al., 1989; Mirgani, 1992; Al-Qarawi, 1999). In the present work we have attempted to extend these studies further by investigating some physiological and biochemical effects of dehydration (10 days) and rehydration, or starvation (7 days) on lipid concentrations in the plasma and erythrocyte membranes of desert camels. For comparative purposes, these experiments were also carried out in normal desert sheep and goats. The aim was to try to explain the unique abilities of camel erythrocytes to resist haemolysis by studying the erythrocyte membrane composition, and to quantify the magnitude of differences between the three species in the above-mentioned aspect.
2. Materials and methods 2.1. Chemicals All reagents used were of the highest purity available, and were obtained from Sigma, St. Louis, MO, USA. 2.2. Animals Six healthy male desert camels (about 200 kg), Najdi sheep (about 30 kg) and desert goats (about 20 kg) were purchased from the local market, and housed at the KSU Animal Farm in shaded pens. The 18 animals were left to acclimatize for 2 weeks prior to the start of the experiment. Water, hay and green lucerne were provided ad libitum, and the animals were also provided with concentrates and mineral blocks.
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2.3. Treatments 2.3.1. Dehydration Water (but not food) was withheld from the camels for 10 days. Blood (10 ml) was withdrawn from the jugular vein of all animals into heparinized tubes before and on the last day of dehydration. 2.3.2. Rehydration After the end of the dehydration period, water was provided to camels and 3 h after rehydration, another blood sample (10 ml) was collected from all animals. 2.4. Measurements Part of the blood collected (4 ml) was centrifuged at 900g for 10 min at 5 C to separate plasma from erythrocytes. The latter was used to prepare ghost erythrocyte cells as follows: After centrifugation of heparinized blood, the packed erythrocytes were washed twice with normal saline (0.9% NaCl). Each time the salt was removed by centrifugation, and the number of erythrocytes was counted in 1 ml in the erythrocyte-salt suspension. To break down the erythrocytes and to remove from it the Hb and obtain the membranes only, distilled water (10 ml) was added to the packed cells, vortexed and centrifuged. This process was repeated three times to ensure the removal of all the Hb in the upper layer. The lower layer consisted of erythrocytes membranes (Folch et al., 1957). The phospholipids in the membranes were isolated using thin layer chromatography (TLC) as described by Kuiper et al. (1971) using known phospholipid standards (obtained from Fischer Scientific, Eden Prairie, MN, USA) to determine the classes of phospholipids present as described by the method of Stewart (1980). Total lipids and cholesterol in erythrocyte membranes were measured by the method of Dodge and Phillips (1967). Fatty acids in the erythrocytes membranes were analysed by Gas-liquid-chromatography (GLC) technique described by Ames (1966). Saponification and methylation of the fatty acids with BCl3 in methanol were as described by Kuiper et al. (1971). The equipment used for analysis was from Packard, Model 7400 (USA) with a flame ionization detector, and the column employed consisted of 15% diethylene glycol succinate as stationary phase on Chromosorb as a support. Peaks were identified with the aid of standard methyl esters of the various fatty acids as supplied by Sigma (St. Louis, MO, USA). Total proteins in the erythrocytes membranes were measured by the spectrophometric method of Lowry et al. (1951).
3. Results Total lipids, phospholipids, cholesterol and proteins in the erythrocyte membrane of hydrated camels, sheep and goats are shown in Table 1. The goats recorded the lower level in total lipids and proteins and were significantly (po0:01) lower than the
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Table 1 Total lipids, phospholipids, cholesterol and proteins in the erythrocytes membrane of normal camel, sheep and goats (mean7SEM)
Total lipids (mg/ml packed cells) Total lipids mg 10 10/cell Cholesterol mmol 10 10/cell Phospholipids mmol 10 10/cell Proteins mg 10 10/cell Proteins:total lipids Cholesterol: phospholipid (molar ratio)
Camel
Sheep
Goats
6.1671.2 1.867023 1.4070.07 1.4570.04 5.6771.2 3.05 0.96
6.1270.92 1.7670.26 1.3270.08 1.5970.02 4.0870.6 2.32 0.83
4.8270.16 1.0470.08 1.3070.02 1.5870.3 1.9570.02 1.88 0.82
po0.01. po0.001.
Table 2 Total lipids, phospholipids, cholesterol and proteins in the erythrocytes membrane of normal, dehydrated, rehydrated and starved camels (mean7SEM)
Total lipids (mg/ml packed cells) Total lipids mg 10 10/cell Cholesterol mmol 10 10/cell Phospholipids mmol 10 10/cell Proteins mg 10 10/cell Proteins:total lipids Choesterol:phospholipid
Normal
Dehydrated
Rehydrated
Starved
6.1671.2 1.8670.23 1.470.07 1.4570.04 5.6771.2 3.05 0.96
6.2770.8 1.7870.6 1.4270.02 1.4670.03 5.8771.1 3.04 0.97
6.2671.3 1.970.27 1.3670.08 1.3970.01 5.2671.3 2.76 0.98
6.1970.9 1.6970.04 1.4270.06 1.3670.02 5.6471.1 3.3 1.04
level in camels and sheep. The cholesterol level was significantly higher (po0:01) in camels when compared with sheep and goats. There was no statistically significant variation in the level of phospholipids in the three species studied. Protein contents of the erythrocyte membrane were significantly lower in sheep (po0:01) and goats (po0:001), however, the proteins to total lipids ratio was higher in the camel (3.05) followed by sheep (2.32) and goats (1.88). Cholesterol to phospholipid ratio was higher in camel than in sheep and goats. The concentration of total lipids, phospholipids, cholesterol, proteins, proteins to total lipid ratio and cholesterol to phospholipid ratio in the erythrocyte membrane of hydrated, dehydrated, rehydrated and starved camels are presented in Table 2. As shown in the table, different treatment did not change the concentration of these constituents significantly. The phospholipid classes in the erythrocyte membrane of hydrated, dehydrated, rehydrated and starved camels are shown in Table 3. Six phospholipid classes were detected in the erythrocyte membrane of camels: phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, phosphatidyl ethanolamine, phosphatidylcholine and sphingomyelin. There was no statistically significant change in the concentration of phospholipid classes during dehydration, rehydration and starvation when compared with hydration.
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Table 3 Phospholipid classes in the erythrocytes membrane of normal, dehydrated, rehydrated and starved camels (mg/mg total lipid-mean7SEM)
Phosphatidyl glycerol Phosphatidyl inositol Phosphatidyl serine Phosphatidyl ethanolamine Phosphatidyl choline Sphingomyelin
Normal
Dehydrated
Rehydrated
Starved
5.8271.40 1.8070.80 3.4670.60 2.2170.06 22.1273.41 65.7474.62
2.7971.40 2.4270.80 4.6271.22 2.6471.10 21.1471.30 66.2175.32
3.6671.12 2.7270.92 3.2270.90 2.9670.80 23.1272.41 64.2173.30
3.2471.17 3.3971.62 4.1271.25 2.8570.60 22.9672.76 63.4474.27
Table 4 Phospholipid classes in the erythrocytes membrane in normal camels, sheep and goats (mg/mg total lipidmean7SEM) Camels Phosphatidyl glycerol Phosphatidyl inositol Phosphatidyl serine Phosphatidyl ethanolamine Phosphatidyl choline Sphingomyelin
5.8271.40 1.8070.80 3.4670.60 2.2170.06 22.1273.41 65.7474.62
Sheep
Goats
11.2471.70 2.7270.08 6.2270.06 3.4670.03 14.3272.41 62.2874.3
14.8673.21 1.5270.04 7.2271.40 3.8670.90 13.2472.74 59.3274.44
po0.01.
The phospholipid classes in the erythrocyte membrane of the hydrated camels, sheep and goats are shown in Table 4. As shown in the table, six phospholipid fractions are present in the erythrocyte membranes of the three species studied. The concentration of phosphatidyglycerol and phosphatidylethanolamine were significantly (po0:01) lower in camels when compared with sheep and goats, while the concentration of shingomyelin was significantly lower in goats than in camels and sheep. There were no significant variation in the concentrations of phosphatidylinositol, phosphatidylcholine in camels, sheep and goats. Fatty acid composition of camel erythrocytes phospholipid were estimated in phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, phosphotidylserine only. The results are presented in Table 5. Each of the phospholipids showed a distinctive acid pattern. It can be noticed that phosphatidylcholine and shingomyelin contain more saturated fatty acids (palmitic and stearic) than the acidic phospholipids (phosphatidylserine and phosphatidylethanolamine). It can be noticed that lignoceric (24:0) and nervonic (24:1) are present exclusively in sphingomyelin.
4. Discussion It is well documented that the erythrocytes of camels are resistant to haemolysis (osmotic fragility is outstandingly low) when compared with sheep, goats, man and
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Table 5 Fatty acid composition of camel erythrocyte major phospholipids Fatty acid
16:0 18:0 18:1 18:2 20:0 20:1 20:2 20:3 20:4 22:0 22:2 22:5 22:6 24:0 24:1
Percentage of total fatty acids Phosphatidyl-choline
Sphingomyelin
Phosphatidyl-ethanolamine
Phosphatidyl-serine
23.6 22.6 18.1 23.1 0.6 0.4 1.2 0.5 7.2 — — — — 2.8 —
14.4 21.3 12.6 2.8 1.4 0.4 1.0 — 5.4 2.4 3.4 — 2.6 23.0 8.3
8.6 14.3 46.4 25.9 1.5 — — 0.5 2.4 — — — — 0.4 —
4.7 27.2 44.7 21.2 0.6 — — — 0.8 — — 0.4 0.2 0.3 —
other animals (Livne and Kuiper, 1973; Mirgani, 1992; Al-Qarawi, 1999). This stability of camel erythrocyte is apparently not due to the morphological characteristics of the erythrocyte alone (Agar and Board, 1983, pp. 55–56). Characteristics such as ability of the erythrocyte to swell to twice their volume in hypotonic media, their resistance to the lytic effect of snake venom (Turner et al., 1958; Condrea et al., 1964) and resistance to sonic haemolysis, indicate some unusual properties of the membrane of the camel erythrocytes. Ralston (1975) found that the major proteins of the camel erythrocyte membranes are similar to those of humans and bovine species, but with a major difference in the major intrinsic membrane water-soluble protein ‘‘spectrin’’ which appears to be very tightly bound to the camel erythrocyte membrane. Concurrent with the total release of spectrin, the camel cells undergo a shape change from flat ellipsoids to spheres, suggesting an important shape-maintaining role for spectrin in the erythrocytes of this species. The available information about these membranes under harsh conditions is limited. This study addresses the lipid composition of erythrocytes of camels subjected to hydration, dehydration, rehydration and starvation. In the present investigation it was found that the concentration of lipids, cholesterol, proteins, proteins to total lipid ratio and cholesterol to phospholipids molar ratio in the erythrocyte membranes of hydrated camels are higher than those of hydrated sheep and goats. It is well known that the contents and ratios of the mentioned constituents are important factors in the stability, life-span and resistance of the erythrocytes to haemolysis. Since the erythrocytes of camels are more resistant to haemolysis, the present findings may lend support to the suggestion that there is a positive correlation between these constituents and the osmotic fragility and stability of erythrocytes. The present results disagree with Cooper (1979) who found that
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increased cholesterol concentration in the membranes in humans causes a decrease in membrane fluidity, and these changes are associated with a reduction in membrane permeability, a distortion of cell contour, filterability and shortening of survival of red cells in vivo. On the other hand, Livne and Kuiper (1973) suggested that the cholesterol to phospholipid ratio is the most important factor because cholesterol is insoluble in water but soluble in phospholipids. In the present study it was found that the cholesterol to phospholipids ratio is higher in camels (0.9) while it was 0.83 and 0.82 in sheep and goats, respectively. As for the concentration of phospholipids in the erythrocyte membrane, it was found that there were no significant variations in their concentration in camels, sheep and goats, indicating that the concentration of phospholipids as such is playing a minor role in the stability of the erythrocytes. Similar conclusions were reported in camels by Livne and Kuiper (1973). However, it was suggested by various workers that the phospholipid classes and not the phospholipid concentrations, are important in the determination of the physiological functions of the erythrocytes (Schwartz et al., 1985; Mirgani, 1992; Warda and Zeisig, 2000). In the present study it was found that the sphingomyelin fraction is the most abundant in all animals studied, followed by phosphatidyl choline. The sum of these two classes constituted 87%, 74% and 72% in camels, sheep and goats, respectively. This percentage is higher than that reported by Mirgani (1992) in camels and goats, and Livne and Kuiper (1973), Al-Qarawi (1999) and Warda and Zeisig (2000) in camels. It worth noting that the percentage of phosphatidyserine and phosphatidylethanolamine is lower in camels when compared with sheep and goats. It was reported by Schwartz et al. (1985) that the phosphatidyl choline and sphingomyelin classes are found in the outer leaflet and the amino phosopholipids (phosphatidylserine and phosphatidylethanolamine) in the cytoplasmic leaflet of the erythrocyte. This arrangement is preferentially maintained by complex, and at present, poorly understood noncovalent interaction between lipids and a skeletal protein, spectrin. Hagve et al. (1993) reported that the ratio of: Spingomyelin+phosphatidylcholine/phosphatidylehanolamineis governing the fragility of erythrocytes. If this ratio decreased, this will result in increased fragility in human erythrocytes. It seems that the same is true for camel erythrocytes because Kuiper et al. (1971) found that in hamsters the osmotic fragility increased when hamsters were exposed to heat and they found that this was associated with lowered content of sphingomyelin and phosphatidylcholine and higher content of phosphatidylserine and phosphatidylethanolamine. The composition of the phospholipid fatty acids, as presented in Table 5, shows that the fatty acid distribution is similar to the distribution in man, cattle, sheep, goats and mice (Dodge and Phillips, 1967; Nelson, 1972; Livne and Kuiper, 1973). This may suggest that the distribution of fatty acids in the phospholipids is not contributing much to the osmotic stability of the erythrocytes. When camels were subjected to dehydration, rehydration and starvation they managed to maintain the concentration of the constituents of the erythrocytic membrane (total lipids, cholesterol, phospholipids, proteins, protein to total lipids and cholesterol to phospholipid ratios) within the hydrated (normal) level.
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It can be concluded that the lower osmotic fragility of the erythrocytes of camel is due, in part, to the high concentration of total lipids, cholesterol, proteins, sphingomyelin and phosphatidylcholine in the erythrocyte membranes of camels when compared with the concentrations of these parameters in the erythrocyte membranes of sheep and goats. The higher ratio of proteins to total lipids; cholesterol to phospholipids in the erythrocyte membranes of camels may have a role in the stability of the camel erythrocytes.
Acknowledgements This work was supported by King Abdulaziz City for Science and Technology.
References Agar, N.S., Board, P.G., 1983. Red Blood Cells of Domestic Mammals, 1st Edition. Elsevier, Amsterdam, 420pp. Ali, K.E., Mousa, H.M., Hume, I.D., 1982. Total body water and water economy in camels, desert goats and desert sheep. Iugoslavica Physiologica et Pharmacologica Acta 18, 221–225. Al-Qarawi, A.A., 1999. Phospholipids of the camel erythrocyte membrane. Journal of Camel Practice and Research 6, 39–40. Ames, B.N., 1966. In: Colowick, S.P., Kaplan, N.D. (Eds.), Methods of Enzymology, Vol. 8. Academic Press, New York, pp. 115–118. Condrea, E., Mammon, Z., Aloof, S., De Vries, A., 1964. Susceptibility of erythrocytes of various animals species to the hemolytic and phospholipid splitting action of snake venoms. Biochimica et Biophysica Acta 84, 365–375. Cooper, R.A., 1979. Influence of increased membrane cholesterol on membrane fluidity and cell function of human red blood cells. Journal of Supramolecular Structure 8, 413–430. Dodge, J.T., Phillips, G.B., 1967. Composition of phospholipids and of phospholipid fatty acids and aldehydes in human red cells. Journal of Lipid Research 8, 667–674. Eitan, A., Aloni, B., Livene, A., 1976. Unique properties of the camel erythrocyte membrane, 11. Organization of the membrane proteins. Biochimica et Biophysica Acta 426, 647–658. Folch, J., Lees, M., Sloane Stanley, G.H., 1957. A simple method for the isolation and purification of total lipids from animal tissues. Journal of Biological Chemistry 226, 497–509. Hagve, T.A., Lie, O., Gronn, M., 1993. The effect of dietary N-3 fatty acids on osmotic fragility and membrane fluidity of human erythrocytes. Scandinavian Journal of Clinical Laboratory Investigation 215, 75–84. Jain, N.C., Keeton, K.S., 1974. Morphology of camel and llama erythrocytes as viewed with scanning electron microscopy. British Veterinary Journal 130, 288–291. Kuiper, P.J.C., Livne, A., Meyerstein, N., 1971. Changes in lipid composition and osmotic fragility of erythrocytes of Hamster induced by heat exposure. Biochimica et Biophysica Acta 248, 300–305. Livne, A., Kuiper, P.J., 1973. Unique properties of the camel erythrocyte membrane. Biochimica et Biophysica Acta 318, 41–49. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin Phenol reagent. Journal of Biological Chemistry 193, 265–275. MacFarlane, W.V., Morris, R.J., Howard, B., 1963. Turn-over and distribution of water in desert camels, sheep and kangaroos. Nature 197, 270–271. Mirgani, T., 1992. Lipid composition of camel erythrocytes. Journal of Arid Environment 22, 401–405.
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Mousa, H.M., Ali, K.E., Hume, I.D., 1983. Effect of water deprivation on urea metabolism in camel, desert sheep and goats. Comparative Biochemistry Physiology 74, 715–720. Nelson, G.J., 1972. In: Nelson, G.J. (Ed.), Blood Lipids and Lipoproteins: Quantitation, Composition and Metabolism. Wiley, New York, NY, pp. 317. Omorphos, S.A., Hawkey, C.M., Rice-Evans, C., 1989. The elliptocyte: a study of the relation between cell shape and membrane structure using the camelid erythrocyte as a model. Comparative Biochemistry Physiology B 94, 789–795. Perk, K., 1963. Haematology: the camel’s erythrocyte. Nature 2000, 272–273. Perk, K., Hort, I., Perri, A., 1964. Osmotic fragility of red blood cells of young and mature domestic and laboratory animals. American Journal of Veterinary Research 25, 1241–1244. Ralston, G.B., 1975. Proteins of the camel erythrocyte membrane. Biochimica et Biophysica Acta 401, 83–94. Schmidt-Nielsen, B., Schmidt Nielsen, K., Houpt, T.R., Jarnum, S.A., 1956. Water balance of the camel. American Journal of Physiolology 185, 185–194. Schwartz, R.S., Chiu, D.T., Lubin, B., 1985. Plasma membrane phospholipid organization in human erythrocyte. Current Tropical Hematology 5, 63–112. Smith, J.E., Mohandas, N., Shohet, S.B., 1979. Variability in erythrocyte deformability among various animals. American Journal of Physiology 236, 725–730. Stewart, J.C.M., 1980. Colorimetric determination of phospholipids with ammonium ferrothiocyanate. Analytical Chemistry 104, 10–14. Turner, J., Anderson, H., Candal, C., 1958. Species difference in red blood cell phosphatides separated by column and paper chromatography. Biochimica et Biophysica Acta 30, 130–134. Warda, M., Zeisig, R., 2000. Phospholipid- and fatty acid-composition in the erythrocyte membrane of the one-humped camel (Camelus dromedaries) its influence on vesicle properties prepared from these lipids. Deutsche Tierarztliche Wochenschrift 107, 368–373.
Lipid concentrations in erythrocyte membranes in normal, starved, dehyrated and rehydrated camels (Camelus dromedarius), and in normal sheep (Ovis aries) and goats (Capra hircus) A.A. Al-Qarawi, H.M. Mousa* Department of Veterinary Medicine, College of Agriculture and Veterinary Medicine, King Saud University, Gaseem Branch, P.O. Box 10158, Buraydah, Al-Gaseem 81999, Saudi Arabia Received 8 January 2003; received in revised form 14 January 2004; accepted 10 February 2004
Abstract The present work compared the effects of starvation (7 days) and dehydration (10 days) followed by rehydration, on erythrocyte membranes of the desert camel (Camelus dromedarius). Total lipids, cholesterol, total phospholipids, phospholipid classes, fatty acids of phospholipid classes and proteins in the erythrocyte membranes of normal camels, sheep (Ovis aries) and desert goats (Capra hircus) have also been investigated. The results suggested that the lipid profile in the erythrocytes of the normal camels was significantly different from those found in normal sheep and goats. Between the two later species no significant differences were observed. In the erythrocyte membranes of the camel two classes of phospholipids (phosphatidylcholine and sphingomyelin), cholesterol and proteins were significantly higher than in sheep and goats. These constituents were not altered significantly by dehydration or starvation in the camel. These results may help to explain the resistance of camel erythrocytes to haemolysis after rehydration, infection with blood parasites or when confronted with other harsh environmental conditions. r 2004 Elsevier Ltd. All rights reserved. Keywords: Camel; Dehydration; Erythrocytes; Sheep; Goats; Saudi Arabia
*Corresponding author. E-mail address: [email protected] (H.M. Mousa). 0140-1963/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.jaridenv.2004.02.004
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1. Introduction Desert camels (Camelus dromedarius) are endowed with certain anatomical, physiological, biochemical and pharmacological peculiarities, which make them distinct from other related ruminants. For example, they are well known for their exceptional ability to withstand long periods of dehydration (MacFarlane et al., 1963; Perk, 1963), their ability to replenish lost water after long periods of dehydration in a relatively short period of time (10 l/min) (Schmidt-Nielsen et al., 1956), and to utilize low-quality roughages (Mousa et al., 1983). It is established that camel erythrocytes are resistant to haemolysis and can expand to about twice its original size when placed under hypotonic solutions (Perk et al., 1964). These properties were partly explained on the basis of their shape as camel erythrocytes are different from mammalian types in being oval, rather than circular discs (Turner et al., 1958; Jain and Keeton, 1974), and partly on their membranes (Livne and Kuiper, 1973; Mirgani, 1992). Several studies have attempted to study the ability of the camel to withstand dehydration and rapid rehydration (e.g. Ali et al., 1982; Mousa et al., 1983), and the contribution of the erythrocytes and their membranes composition to the physiological adaptation to dehydration (Livne and Kuiper, 1973; Eitan et al., 1976; Smith et al., 1979; Omorphos et al., 1989; Mirgani, 1992; Al-Qarawi, 1999). In the present work we have attempted to extend these studies further by investigating some physiological and biochemical effects of dehydration (10 days) and rehydration, or starvation (7 days) on lipid concentrations in the plasma and erythrocyte membranes of desert camels. For comparative purposes, these experiments were also carried out in normal desert sheep and goats. The aim was to try to explain the unique abilities of camel erythrocytes to resist haemolysis by studying the erythrocyte membrane composition, and to quantify the magnitude of differences between the three species in the above-mentioned aspect.
2. Materials and methods 2.1. Chemicals All reagents used were of the highest purity available, and were obtained from Sigma, St. Louis, MO, USA. 2.2. Animals Six healthy male desert camels (about 200 kg), Najdi sheep (about 30 kg) and desert goats (about 20 kg) were purchased from the local market, and housed at the KSU Animal Farm in shaded pens. The 18 animals were left to acclimatize for 2 weeks prior to the start of the experiment. Water, hay and green lucerne were provided ad libitum, and the animals were also provided with concentrates and mineral blocks.
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2.3. Treatments 2.3.1. Dehydration Water (but not food) was withheld from the camels for 10 days. Blood (10 ml) was withdrawn from the jugular vein of all animals into heparinized tubes before and on the last day of dehydration. 2.3.2. Rehydration After the end of the dehydration period, water was provided to camels and 3 h after rehydration, another blood sample (10 ml) was collected from all animals. 2.4. Measurements Part of the blood collected (4 ml) was centrifuged at 900g for 10 min at 5 C to separate plasma from erythrocytes. The latter was used to prepare ghost erythrocyte cells as follows: After centrifugation of heparinized blood, the packed erythrocytes were washed twice with normal saline (0.9% NaCl). Each time the salt was removed by centrifugation, and the number of erythrocytes was counted in 1 ml in the erythrocyte-salt suspension. To break down the erythrocytes and to remove from it the Hb and obtain the membranes only, distilled water (10 ml) was added to the packed cells, vortexed and centrifuged. This process was repeated three times to ensure the removal of all the Hb in the upper layer. The lower layer consisted of erythrocytes membranes (Folch et al., 1957). The phospholipids in the membranes were isolated using thin layer chromatography (TLC) as described by Kuiper et al. (1971) using known phospholipid standards (obtained from Fischer Scientific, Eden Prairie, MN, USA) to determine the classes of phospholipids present as described by the method of Stewart (1980). Total lipids and cholesterol in erythrocyte membranes were measured by the method of Dodge and Phillips (1967). Fatty acids in the erythrocytes membranes were analysed by Gas-liquid-chromatography (GLC) technique described by Ames (1966). Saponification and methylation of the fatty acids with BCl3 in methanol were as described by Kuiper et al. (1971). The equipment used for analysis was from Packard, Model 7400 (USA) with a flame ionization detector, and the column employed consisted of 15% diethylene glycol succinate as stationary phase on Chromosorb as a support. Peaks were identified with the aid of standard methyl esters of the various fatty acids as supplied by Sigma (St. Louis, MO, USA). Total proteins in the erythrocytes membranes were measured by the spectrophometric method of Lowry et al. (1951).
3. Results Total lipids, phospholipids, cholesterol and proteins in the erythrocyte membrane of hydrated camels, sheep and goats are shown in Table 1. The goats recorded the lower level in total lipids and proteins and were significantly (po0:01) lower than the
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Table 1 Total lipids, phospholipids, cholesterol and proteins in the erythrocytes membrane of normal camel, sheep and goats (mean7SEM)
Total lipids (mg/ml packed cells) Total lipids mg 10 10/cell Cholesterol mmol 10 10/cell Phospholipids mmol 10 10/cell Proteins mg 10 10/cell Proteins:total lipids Cholesterol: phospholipid (molar ratio)
Camel
Sheep
Goats
6.1671.2 1.867023 1.4070.07 1.4570.04 5.6771.2 3.05 0.96
6.1270.92 1.7670.26 1.3270.08 1.5970.02 4.0870.6 2.32 0.83
4.8270.16 1.0470.08 1.3070.02 1.5870.3 1.9570.02 1.88 0.82
po0.01. po0.001.
Table 2 Total lipids, phospholipids, cholesterol and proteins in the erythrocytes membrane of normal, dehydrated, rehydrated and starved camels (mean7SEM)
Total lipids (mg/ml packed cells) Total lipids mg 10 10/cell Cholesterol mmol 10 10/cell Phospholipids mmol 10 10/cell Proteins mg 10 10/cell Proteins:total lipids Choesterol:phospholipid
Normal
Dehydrated
Rehydrated
Starved
6.1671.2 1.8670.23 1.470.07 1.4570.04 5.6771.2 3.05 0.96
6.2770.8 1.7870.6 1.4270.02 1.4670.03 5.8771.1 3.04 0.97
6.2671.3 1.970.27 1.3670.08 1.3970.01 5.2671.3 2.76 0.98
6.1970.9 1.6970.04 1.4270.06 1.3670.02 5.6471.1 3.3 1.04
level in camels and sheep. The cholesterol level was significantly higher (po0:01) in camels when compared with sheep and goats. There was no statistically significant variation in the level of phospholipids in the three species studied. Protein contents of the erythrocyte membrane were significantly lower in sheep (po0:01) and goats (po0:001), however, the proteins to total lipids ratio was higher in the camel (3.05) followed by sheep (2.32) and goats (1.88). Cholesterol to phospholipid ratio was higher in camel than in sheep and goats. The concentration of total lipids, phospholipids, cholesterol, proteins, proteins to total lipid ratio and cholesterol to phospholipid ratio in the erythrocyte membrane of hydrated, dehydrated, rehydrated and starved camels are presented in Table 2. As shown in the table, different treatment did not change the concentration of these constituents significantly. The phospholipid classes in the erythrocyte membrane of hydrated, dehydrated, rehydrated and starved camels are shown in Table 3. Six phospholipid classes were detected in the erythrocyte membrane of camels: phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, phosphatidyl ethanolamine, phosphatidylcholine and sphingomyelin. There was no statistically significant change in the concentration of phospholipid classes during dehydration, rehydration and starvation when compared with hydration.
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Table 3 Phospholipid classes in the erythrocytes membrane of normal, dehydrated, rehydrated and starved camels (mg/mg total lipid-mean7SEM)
Phosphatidyl glycerol Phosphatidyl inositol Phosphatidyl serine Phosphatidyl ethanolamine Phosphatidyl choline Sphingomyelin
Normal
Dehydrated
Rehydrated
Starved
5.8271.40 1.8070.80 3.4670.60 2.2170.06 22.1273.41 65.7474.62
2.7971.40 2.4270.80 4.6271.22 2.6471.10 21.1471.30 66.2175.32
3.6671.12 2.7270.92 3.2270.90 2.9670.80 23.1272.41 64.2173.30
3.2471.17 3.3971.62 4.1271.25 2.8570.60 22.9672.76 63.4474.27
Table 4 Phospholipid classes in the erythrocytes membrane in normal camels, sheep and goats (mg/mg total lipidmean7SEM) Camels Phosphatidyl glycerol Phosphatidyl inositol Phosphatidyl serine Phosphatidyl ethanolamine Phosphatidyl choline Sphingomyelin
5.8271.40 1.8070.80 3.4670.60 2.2170.06 22.1273.41 65.7474.62
Sheep
Goats
11.2471.70 2.7270.08 6.2270.06 3.4670.03 14.3272.41 62.2874.3
14.8673.21 1.5270.04 7.2271.40 3.8670.90 13.2472.74 59.3274.44
po0.01.
The phospholipid classes in the erythrocyte membrane of the hydrated camels, sheep and goats are shown in Table 4. As shown in the table, six phospholipid fractions are present in the erythrocyte membranes of the three species studied. The concentration of phosphatidyglycerol and phosphatidylethanolamine were significantly (po0:01) lower in camels when compared with sheep and goats, while the concentration of shingomyelin was significantly lower in goats than in camels and sheep. There were no significant variation in the concentrations of phosphatidylinositol, phosphatidylcholine in camels, sheep and goats. Fatty acid composition of camel erythrocytes phospholipid were estimated in phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, phosphotidylserine only. The results are presented in Table 5. Each of the phospholipids showed a distinctive acid pattern. It can be noticed that phosphatidylcholine and shingomyelin contain more saturated fatty acids (palmitic and stearic) than the acidic phospholipids (phosphatidylserine and phosphatidylethanolamine). It can be noticed that lignoceric (24:0) and nervonic (24:1) are present exclusively in sphingomyelin.
4. Discussion It is well documented that the erythrocytes of camels are resistant to haemolysis (osmotic fragility is outstandingly low) when compared with sheep, goats, man and
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Table 5 Fatty acid composition of camel erythrocyte major phospholipids Fatty acid
16:0 18:0 18:1 18:2 20:0 20:1 20:2 20:3 20:4 22:0 22:2 22:5 22:6 24:0 24:1
Percentage of total fatty acids Phosphatidyl-choline
Sphingomyelin
Phosphatidyl-ethanolamine
Phosphatidyl-serine
23.6 22.6 18.1 23.1 0.6 0.4 1.2 0.5 7.2 — — — — 2.8 —
14.4 21.3 12.6 2.8 1.4 0.4 1.0 — 5.4 2.4 3.4 — 2.6 23.0 8.3
8.6 14.3 46.4 25.9 1.5 — — 0.5 2.4 — — — — 0.4 —
4.7 27.2 44.7 21.2 0.6 — — — 0.8 — — 0.4 0.2 0.3 —
other animals (Livne and Kuiper, 1973; Mirgani, 1992; Al-Qarawi, 1999). This stability of camel erythrocyte is apparently not due to the morphological characteristics of the erythrocyte alone (Agar and Board, 1983, pp. 55–56). Characteristics such as ability of the erythrocyte to swell to twice their volume in hypotonic media, their resistance to the lytic effect of snake venom (Turner et al., 1958; Condrea et al., 1964) and resistance to sonic haemolysis, indicate some unusual properties of the membrane of the camel erythrocytes. Ralston (1975) found that the major proteins of the camel erythrocyte membranes are similar to those of humans and bovine species, but with a major difference in the major intrinsic membrane water-soluble protein ‘‘spectrin’’ which appears to be very tightly bound to the camel erythrocyte membrane. Concurrent with the total release of spectrin, the camel cells undergo a shape change from flat ellipsoids to spheres, suggesting an important shape-maintaining role for spectrin in the erythrocytes of this species. The available information about these membranes under harsh conditions is limited. This study addresses the lipid composition of erythrocytes of camels subjected to hydration, dehydration, rehydration and starvation. In the present investigation it was found that the concentration of lipids, cholesterol, proteins, proteins to total lipid ratio and cholesterol to phospholipids molar ratio in the erythrocyte membranes of hydrated camels are higher than those of hydrated sheep and goats. It is well known that the contents and ratios of the mentioned constituents are important factors in the stability, life-span and resistance of the erythrocytes to haemolysis. Since the erythrocytes of camels are more resistant to haemolysis, the present findings may lend support to the suggestion that there is a positive correlation between these constituents and the osmotic fragility and stability of erythrocytes. The present results disagree with Cooper (1979) who found that
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increased cholesterol concentration in the membranes in humans causes a decrease in membrane fluidity, and these changes are associated with a reduction in membrane permeability, a distortion of cell contour, filterability and shortening of survival of red cells in vivo. On the other hand, Livne and Kuiper (1973) suggested that the cholesterol to phospholipid ratio is the most important factor because cholesterol is insoluble in water but soluble in phospholipids. In the present study it was found that the cholesterol to phospholipids ratio is higher in camels (0.9) while it was 0.83 and 0.82 in sheep and goats, respectively. As for the concentration of phospholipids in the erythrocyte membrane, it was found that there were no significant variations in their concentration in camels, sheep and goats, indicating that the concentration of phospholipids as such is playing a minor role in the stability of the erythrocytes. Similar conclusions were reported in camels by Livne and Kuiper (1973). However, it was suggested by various workers that the phospholipid classes and not the phospholipid concentrations, are important in the determination of the physiological functions of the erythrocytes (Schwartz et al., 1985; Mirgani, 1992; Warda and Zeisig, 2000). In the present study it was found that the sphingomyelin fraction is the most abundant in all animals studied, followed by phosphatidyl choline. The sum of these two classes constituted 87%, 74% and 72% in camels, sheep and goats, respectively. This percentage is higher than that reported by Mirgani (1992) in camels and goats, and Livne and Kuiper (1973), Al-Qarawi (1999) and Warda and Zeisig (2000) in camels. It worth noting that the percentage of phosphatidyserine and phosphatidylethanolamine is lower in camels when compared with sheep and goats. It was reported by Schwartz et al. (1985) that the phosphatidyl choline and sphingomyelin classes are found in the outer leaflet and the amino phosopholipids (phosphatidylserine and phosphatidylethanolamine) in the cytoplasmic leaflet of the erythrocyte. This arrangement is preferentially maintained by complex, and at present, poorly understood noncovalent interaction between lipids and a skeletal protein, spectrin. Hagve et al. (1993) reported that the ratio of: Spingomyelin+phosphatidylcholine/phosphatidylehanolamineis governing the fragility of erythrocytes. If this ratio decreased, this will result in increased fragility in human erythrocytes. It seems that the same is true for camel erythrocytes because Kuiper et al. (1971) found that in hamsters the osmotic fragility increased when hamsters were exposed to heat and they found that this was associated with lowered content of sphingomyelin and phosphatidylcholine and higher content of phosphatidylserine and phosphatidylethanolamine. The composition of the phospholipid fatty acids, as presented in Table 5, shows that the fatty acid distribution is similar to the distribution in man, cattle, sheep, goats and mice (Dodge and Phillips, 1967; Nelson, 1972; Livne and Kuiper, 1973). This may suggest that the distribution of fatty acids in the phospholipids is not contributing much to the osmotic stability of the erythrocytes. When camels were subjected to dehydration, rehydration and starvation they managed to maintain the concentration of the constituents of the erythrocytic membrane (total lipids, cholesterol, phospholipids, proteins, protein to total lipids and cholesterol to phospholipid ratios) within the hydrated (normal) level.
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It can be concluded that the lower osmotic fragility of the erythrocytes of camel is due, in part, to the high concentration of total lipids, cholesterol, proteins, sphingomyelin and phosphatidylcholine in the erythrocyte membranes of camels when compared with the concentrations of these parameters in the erythrocyte membranes of sheep and goats. The higher ratio of proteins to total lipids; cholesterol to phospholipids in the erythrocyte membranes of camels may have a role in the stability of the camel erythrocytes.
Acknowledgements This work was supported by King Abdulaziz City for Science and Technology.
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