Plasma Lipids and Lipoproteins of Some Members of the Order Perissodactyla
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Comp. Biochem. Ph~'siol., Wol. 63B, pp. 275 to 281 0305-0491/79/0601 0275502.00,0 ,i, Pergamon Press Ltd 1979. Printed in Great Britain PLASMA LIPIDS AND LIPOPROTEINS OF SOME MEMBERS OF THE ORDER PERISSODACTYLA W. M. F. LEAT, CHRISTINE A. NORTHROP, N. BUTTRESS and D. M. JONES* Agricultural Research Council, Institute of Animal Physiology, Babraham, Cambridge CB2 4AT and *Zoological Society of London, Regents Park, London, NWI, U.K. (Received 9 October 1978) Abstract--l. The plasma lipoproteins of various members of the order Perissodactyla have been exam- ined by electrophoresis and analytical ultracentrifugation. 2. In the Equidae, high density (ct) lipoprotein was the major component (80-90~o) and low density (fl) lipoprotein (10-20%) the minor component. 3. In the Tapiridae represented by the Malayan tapir (Tapirus indicus), high density and low density lipoproteins were present in approximately equal amounts. 4. In the Rhinocerotidae, the high density lipoprotein characteristic of the Equidae and Tapiridae was absent, and the plasma lipoproteins consisted of a complex group having fl mobility on electro- phoresis and a flotation pattern usually associated with low density lipoprotein. 5. The fatty acid composition of plasma lipids was remarkably similar in all members of the Perisso- dactyla examined, with very high percentages of linoleic acid (> 70°/0) being found in the cholesteryl esters. INTRODUCTION nary observations (Leat et al., 1975) indicated that there was no obvious relationship between the mode In herbivorous animals the mode of digestion of diet- of lipid digestion and the profile of plasma lipopro- ary lipid is dependent on the anatomy and physiology teins, but interesting differences were noted between of the digestive tract. In simple stomached animals members of differing orders. The plasma lipoproteins such as the horse (Equus caballus) and pig (Sus scrofa), of some members of the order Perissodactyla are now lipid is presumably digested in the small intestine in reported here in more detail. a manner similar to that seen in man (Homo sapiens) and the rat (Rattus norvegicus), whereas in herbivores with complex stomachs, such as the domestic ox (Bos MATERIALS AND METHODS taurus) and sheep (Ovis aries) extensive hydrolysis of dietary lipids occurs anterior to the small intestine Animals in the rumen (see Garton, 1967). In addition, the size The non-domestic perissodactyl ungulates (see Fig. 1) of lipid droplets absorbed into the lymphatics of were maintained by the Zoological Society of London ruminant animals appears to be .smaller than that either at Regents Park or Whipsnade Park. Blood samples seen in non-ruminants, which could affect the sub- were taken by venepuncture from animals which had been sequent metabolism of the absorbed lipids (see Harri- sedated for veterinary examination or movement to son &Leat, 1975). To investigate whether differences another enclosure. Blood was collected in tubes containing between ruminant and non-ruminant animals in the thiomersal (0.1 mg/ml blood) and EDTA (1 mg/ml blood). and centrifuged to obtain the plasma which was then digestion and absorption of lipids had any effect on stored at -20°C until analysis. the subsequent mode of transport of lipid in plasma, The Equidae examined were fed on a diet of meadow the distribution of plasma lipoproteins in domestic hay and commercial horse cubes. The white rhinoceros and non-domestic herbivores was examined. Prelimi- (Ceratotherium simum) were fed similarly, but with the ORDER PE~ISSODACTYLA SUBORD~ HIPPOMORPHA ~"~oMo~ SUP~FAMILY EQU01DEA TAPIROIDEAJ\ RHINOC~OII~A FAMILY EQIJID~ RH!I~0T I JibE ! SPECIES Domestic Horse ~Equus caballus) Malay! Tapir White rbiSnoceros (Ceratoth@rium (Tapirus indicus) Bimum) Wild Horse (Equus przewalski) Black rhinoceros (Diceros bicornis) Common Zebra (Equus burchelli) Indian rhinoceros (Rhinoceros Mountain Zebra (Equus zebra) unico~dis) Donkey (Equus asinus) Onager (Asinus hemlonus) Fig. 1. Classification of the order Perissodactyla. 275 276 W.M.F. LEAT et al. addition of clover or lucerne hay in the winter. The other lipoprotein by 1.26 and the 7 (high density) lipoprotein species sampled received a higher crude protein intake in by 1.83, factors which reflect the relative content of lipid the form of a high protein horse cube or a dairy cube in ovine plasma lipoproteins (Leat et al., 1976). with clover hay. Most of the animals kept at Whipsnade The total lipoproteins of plasma were separated by cen- have access to grass in the summer. Those at London trifugation at density 1.21 g/ml for 40 hr at 40,000 rev/min receive fresh vegetables. All animals are basically fed a diet using a 40.3 rotor in a Beckman preparative ultracentrifuge suitable for maintenance only. Blood samples from the (Model L2 65B). The lipoproteins were removed with a domestic horse and donkey (Equus asinus) were obtained fine Pasteur pipette (De Lalla & Gofman, 1954) and dia- through local veterinary practice. lysed against buffer of density 1.21 g/ml. Only samples from adult animals which were in good Analytical ultracentrifugation was carried out in a Beck- health or with minor ailments were analysed. Most of the man Model E Analytical Ultracentrifuge at density samples were from non lactating females. 1.21 g/ml using Schlieren optics. Pairs of samples were examined simultaneously using double sector standard and Analytical wedge cells. Samples were centrifuged at 20 C, first at The plasma lipids were extracted into chloroform meth- 36,000 rpm with photographs being taken every 8 min for anol (2: 1, v/v) and after separation by thin layer chroma- 40min. The speed was then increased to 56,000 rpm and tography the individual lipids were estimated by methods a similar photographic procedure repeated. described by Leat et al. (1976). The fatty acid compositions A second estimate of the percentage composition of the of the individual plasma lipids were determined as de- plasma lipoproteins was obtained by projecting suitable scribed by Bowyer et al. (1964). frames of the Schlieren negatives at a 5-fold magnificalion Electrophoresis of the plasma lipoproteins was carried onto good quality paper. The peaks were marked on the out on cellulose acetate strips basically as described by paper, cut out, weighed and expressed as a percentage of Magnani & Howard (1972). The strips were divided longi- the total. The frames were selected such that the peaks tudinally, one half being stained for protein with amido measured were at similar distances from the base of the black and the other half for lipid by the ozone/Schiff's cell to minimize errors due to radial concentration. reaction. The electrophoretographs were scanned in a den- sitometer with an automatic integrator (Vitatron Scientific Instruments) and figures for areas representing the various lipoproteins were recorded. Estimates of the percentage RES U LTS distribution of the individual lipoproteins were obtained by multiplying the area printout of the fl (low density) Cellulose acetate electrophoresis Some representative tracings of plasma lipoproteins superimposed on the protein separation are shown * The term ~-Iipoprotein is used in electrophoresis for in Fig. 2. In the family Equidae illustrated by the the lipoprotein migrating in the a-globulin region and cor- common zebra (Equus burchelli) and horse (Fig. 2a responds to the high density lipoprotein (HDL) fraction and b) the ~ lipoprotein* migrating in the or-globulin separated by ultracentrifugation. The /3-1ipoprotein mi- grates with the/~-globulin and corresponds to the low den- region just behind the albumen band is the major sity lipoprotein (LDL). The pre-/~ band corresponds to very component comprising 80-90% of the total lipopro- low density lipoprotein (VLDL). teins. The lipoprotein migrating in the /~ globulin M I q I~- aibumen I I I ~HOL II i] nr I I1 !l III It I II #l Ill 11 " 14l IH i I III 171/ " ~H I ~. It~11:, I I,, t ! IIP, t I ilr, :i Ill II illI.! I ^ II It ?t I | ~1 II.:" I I II I ~" '1 ~ I ',i "k t[1 ' ll l, I !1 VLDL II'J / j,i , 1 /\ )jvv /. o o (a) (b) (c] (d] Fig. 2. Cellulose acetate electrophoresis of the plasma of (a) common zebra, (b) domestic horse, (c) white rhinoceros, (d) Malayan tapir. Protein ( ..... ); lipid ( ); © = origin. Plasma lipids and lipoproteins in Perissodactyla 277 IL. Przewalski horse r Domestic horse Common zebra Onager Malay tapir Black rhinoceros Man White rhinoceros White rhinoceros Indian rhinoceros A B Fig. 3. Selected Schlieren patterns from the analytical ultracentrifugation of total plasma lipoproteins of various perissodactyls. Flotation is from right to left at density 1.21 g/ml (see text for further details). A, 24 min at 36,000 rev/min showing separation of low density lipoprotein from the high density lipopro- tein, which remains near the base of the cell (at right). B, 40 min at 56,000 rev/min showing flotation of high density lipoprotein. The low density lipoproteins are now concentrated at the meniscus (left). 278 W. M. F. LEAT et al. Table l. Percentage low density (fl) and high density (~t) lipoproteins in the plasma of various perisso- dactyls (number of animals in brackets) Cellulose acetate Analytical electrophoresis Ultracentrifuge Species lipoprotein ~ lipoprotein LDL Domestic Horse (2) 29.3 70.7 (2) 18.9 81.1 Przewalski horse (1) 15.7 84.3 (1) 21.9 78.1 Ona~r (1) 21.5 78.5 (2) 15.8 84.2 Mountain Zebra (2) 24.1 75.9 (2) 8.1 91.9 Common Zebra (2) 21.3 78.7 (2) 9.5 90.5 White Rhinoceros (5) lOO - (3) lOO - Black Rhinoceros (1) 100 - (1) 100 - Indian Rhinoceros (1) 100 - (1) 100 - Malayan ~apir (2) 42.7 57.3 (2) 41.3 58.7 region is obviously of multiple composition and prob- absence of the HDL characteristic of the Equidae.