Gofman, Jones, Lindgren, Lyon, Elliott and Strisower, 1950; Lewis, post-absorptive state.

Transcription

1 THE PROTEIN AND LIPID COMPOSITION OF THE PLASMA OF DIFFERENT ANIMAL SPECIES DETERMINED BY ZONE ELECTROPHORESIS AND CHEMICAL ANALYSIS. By BEDE MORRIS 1 and F. C. COURTICE. From the Kanematsu Memorial Institute of Pathology, Sydney Hospital, Sydney. THE existence of plasma protein-lipid complexes has been recognized since 1929 when Machebceuf isolated from horse serum a protein containing about 40 per cent lipid. The emphasis placed in recent years on the significance of the blood lipids in arteriosclerosis has led to extensive investigations on the physical state in which they exist in the plasma. Techniques of ultracentrifugation and electrophoresis have revealed the existence of different protein-lipid associations in the plasma of both man and animals [Gurd, Oncley, Edsall and Cohn, 1949; Gofman, Jones, Lindgren, Lyon, Elliott and Strisower, 1950; Lewis, Green and Page, 1952; Oncley and Gurd, 1953; Swahn, 1953]. Many early workers [cf. Bloor, 1943] reported lipid values for the plasma of several different animal species using a variety of techniques for chemical estimation. In view of our own interest in the fundamental problem of lipid transfer across the capillary wall, we have attempted to define the plasma protein and lipid composition of various animals existing under widely different dietary conditions. METHODS Determinations of the protein and lipid fractions in the plasma of man, dog, cat, rat, mouse, ox, goat, sheep, horse, rabbit and guinea-pig were carried out by zone electrophoresis and by chemical analysis. The animals examined varied in age and were of both sexes. The human group consisted of healthy male and female subjects between the ages of 20 and 40 years. All plasma samples were collected by venepuncture, using powdered heparin (Evans) as an anticoagulant. With the exception of the herbivorous animals, the subjects examined were in the post-absorptive state. Zone Electrophoresis.-This was conducted on plasma samples with filter paper as a supporting medium. The apparatus used was similar to that described by Flynn and de Mayo [1951]. Separations were made in a barbiturate buffer of ph 8-6, ionic strength 0-06 M, for 171 hrs. at a potential of 150 V. Whatman No. 1 chromatographic paper was cut 1 This work was carried out with the aid of a grant from the National Health and Medical Research Council, Canberra. VOL. XL, NO

2 128 Morris and Courtice into 3-inch-wide strips and plasma spots applied by micropipette, one to each half of the strip. At the end of each run the strips were dried in a hot-air oven and cut in two, one half being stained for protein with bromophenol blue, and the other for lipid with Sudan black. The drying, staining and washing procedures and the staining materials used were those described by Swahn [1952]. Typical patterns obtained MAN CAT PROTEIN LIPID PROTEIN LIPID FIG. 1.-Typical electrophoretic patterns for human and cat plasma, stained for protein (bromophenol blue) and for lipid (Sudan black). The cross lines represent the points of application of the plasma on the filter paper. in samples of human and cat plasma are shown in fig. 1. The distribution of both protein and fat stains along the strips were measured, after elution, in a Beckman spectrophotometer (see fig. 2). No attempt was made to correct for differences in dye binding capacity by the use of correction factors. The localization of cholesterol on the electrophoretic patterns was determined by spraying with a solution of antimony pentachloride in chloroform and noting the position of the brown colour which developed. Chemical Analysis.-Total protein was estimated by micro-kjeldahl digestion with subsequent Nesslerization. "Albumin" values were

3 Animal Plasma Proteins and Lipids 129 obtained from plasma filtrates after precipitation with 21 per cent sodium sulphite [Campbell and Hanna, 1937]. Non-protein nitrogen was determined on filtrates after protein precipitation with 5 per cent trichloracetic acid. "Globulin" was calculated by subtraction of "albumin" values from total protein. Total esterified fatty acid was measured by the method of Sterne and Shapiro [1953]. Total cholesterol estimations were carried out by direct chloroform extraction, using the method of Kingsley and Schaffert [1949], colour development being achieved with acetic anhydridesulphuric acid reagent. Phospholipid phosphorus was measured by a micro-modification of King's method [1932]. The optical densities of these colorimetric reactions were recorded at the appropriate wavelength on a Beckman D.U. quartz spectrophotometer. All mean values given are accompanied by their standard errors. RESULTS Typical electrophoretic patterns of plasma samples from different species of animals are illustrated in figs. 2 and 3. Human.-The protein and lipid patterns obtained from normal human plasma agreed closely with the results of other workers [Rosenberg, 1952; Kunkel and Slater, 1952; Swahn, 1953]. Good separation was achieved on filter paper, the proteins resolving into characteristic fibrinogen, gamma, beta, alpha2 and alpha, globulins and albumin fractions. The lipoproteins of human plasma were present in relatively high concentration and separated readily into three distinct components. The first represented lipid migrating between the origin and the betaglobulin zone and varied considerably in amount between samples. This component reflected the amount of particulate lipid present in the plasma and was minimal in the post-absorptive state. The second lipid fraction was a distinct area, which stained deeply with Sudan black and was localized with the beta globulin. The third area showed the greatest electrophoretic mobility and extended through the alpha,- globulin area into the albumin zone. This zone always contained much less lipid-staining material than the large beta-lipoprotein zone. Dog.-The protein fractions of dog plasma in general separated into fibrinogen, gamma, beta and alpha globulins and albumin. The fibrinogen peak often failed to separate distinctly from the gamma globulin and in some samples the alpha globulins separated into two peaks. The lipids migrated readily and in the post-absorptive animal two zones could be identified. The first peak contained only a small amount of the plasma lipoprotein and migrated with the beta globulins. The second peak showed a greater electrophoretic mobility and migrated as a well-defined zone extending from the alpha-globulin area into the albumin zone. The concentration of lipoprotein in the samples of

4 130 Morris and Courtice plasma examined was high, and most of it was present in the form of this fast migrating component. In some samples a small zone of lipid was found at the origin of the pattern, but this was variable and attributed MAN DOG CAT RAT MOUSE FIG. 2.-Elution diagrams of electrophoretic patterns of plasma from various species of omnivora. : protein.....: lipid. to the presence of small amounts of absorbed fat present in the plasma in particulate form. Cat.-The protein pattern of cat plasma was characterized by multiple globulin components. As many as two beta and three alpha components could be identified in most samples. The alpha globulins

5 Animal Plasma Proteins and Lipids were present in higher concentrations than the beta globulins. The gamma globulin was characteristically large in all samples studied and was mainly responsible for a relatively low A/G ratio. The lipid patterns showed two areas of staining. The first was localized around the origin ox SHEEP GOAT 131 FIG. 3.-Elution diagrams of electrophoretic patterns of plasma from several species of herbivora. : protein.... : lipid. and varied in intensity with the digestive state; in some post-absorptive samples this zone was almost negligible. The second area stained intensely and corresponded to the faster migrating alpha globulin. This lipoprotein showed a characteristic horning around the edges of the albumin spot. Rat.-Samples showed considerable variation in the protein pattern obtained. There were usually gamma globulin and fibrinogen, beta,

6 132 Morris and Courtice alpha, and alpha, globulins and albumin present. In some samples the alpha globulins showed poor separation. The albumin spot showed the presence of a faster migrating component in several samples examined. The beta-globulin zone was distinct and present in relatively large amounts, whilst the faster migrating alpha1 globulin was present in higher concentration than the alpha2 fraction. The lipids of rat plasma were present in low concentration and electrophoretic separation was poor. A lipid trail extended from the point of application to the albumin zone, where an appreciable amount of lipid was localized. A small peak was present at the beginning of the albumin zone, whilst in those protein patterns which showed a tailing of the albumin spot, the lipid pattern extended right through the albumin zone to form an area of lipoprotein migrating ahead of the main albumin spot. The amount of lipid stain present in any pattern was small and indicated a low level of plasma lipoprotein. This was substantiated by chemical analysis. MoUse.-The protein pattern of the mouse was similar to that of the rat. All fractions showed good separation on filter paper, and could be defined as gamma globulin, fibrinogen, beta, alpha2 and alpha, globulins, and albumin. An additional component, designated as pre-albumin, with an electrophoretic mobility greater than that of the main albumin group, was also present in all samples. The beta-globulin peak was large, as in the rat, whilst the alpha1 globulin was present in greater concentration than the slower migrating alpha2 component (fig. 4). The lipids of mouse plasma separated into three distinct areas. The first zone was localized around the point of application; the second area was ill defined and migrated slightly in advance of the beta-globulin area. The third area was characteristic of mouse plasma and represented a zone of deeply staining lipid material localized in advance of the albumin zone. This area corresponded exactly to the pre-albumin protein area and represented a lipoprotein of high electrophoretic mobility containing a large proportion of the plasma lipids. This lipoprotein was a constant feature of all mouse plasmas examined. Ox.-The protein patterns of the ox showed good separation with fibrinogen, gamma, beta and alpha globulins and albumin. The amount of globulins present in the plasma was high, whilst the A/G ratio was correspondingly low. The lipids migrated into two distinct areas. The first zone, localized around the beta-globulin protein area, was poorly defined. The second zone migrated just behind the albumin spot and contained a relatively large proportion of the plasma lipids. This lipoprotein was constantly present in about the same concentration as the more slowly migrating lipid component. In some samples there was a localization of lipid around the point of application of the plasma.. Sheep.-The protein patterns of the sheep showed poor resolution on filter paper. Only one globulin zone could be established between the gamma globulin-fibrinogen peak and the albumin. This zone was

7 Animal Plasma Proteins and Lipids 133 distinct, and migrated midway between the origin and the albumin zone. Lipid electrophoretic patterns were similar to those of the ox, with a slow migrating peak extending a short distance from the origin, and a faster one which was localized just behind the albumin zone. The concentration of lipoprotein in the plasma was low. PROTEIN LIPID FIG. 4.-Typical electrophoretic pattern of mouse plasma stained for protein (bromophenol blue) and for lipid (Sudan black). Goat.-Only one sample of goat plasma was examined, but this showed good separation on electrophoresis. There was a high gamma globulin and fibrinogen content in the sample, with beta and alpha globulins and albumin fractions. The lipid pattern was typical of those seen for samples of ox and sheep plasma, with two characteristic peaks of about equal size. The amount of lipoprotein present was small. Horse.-The proteins separated into single gamma, beta and alpha globulin zones and a low albumin peak. Lipid patterns showed good

8 134 Morris and Courtice separation, and localization into two fractions was quite distinct. The concentration of lipoprotein in horse plasma was higher than that found in the ox, sheep and goat. The slower migrating component was slightly larger and migrated with the beta-globulin fraction, whilst the faster moving component localized in the alpha globulin-albumin area. There was a small amount of lipid material present at the origin of most patterns. Rabbit.-Protein patterns showed good separation, and fibrinogen, gamma, beta, alpha2 and alpha, globulins and albumin fractions could be identified. The beta peak was larger than either of the alpha globulins. The alpha globulins sometimes failed to separate distinctly into two fractions. The gamma-globulin peak was small, whilst the TABLE I.-Tar PROTEIN AND LIPID CONCENTRATIONS IN THE PLASMA OF VARIous ANIMAL SPECIES The mean results are given together with their standard errors. The figures in brackets indicate the number of plasmas examined. Total Albumin Globulin' Total Total Phospholipid protein, gpecetgprcntfatty acid, cholesterol, phosphorus, g. Per Cent g. per Cent g. Per Cent meq./l. mg. per cent mg. per cent Human (10) 6.85 ± ± ± *1 ± ± ±0.5 Dog (6) 6 20 ± ± ± ± ±1-4 Cat (20) 7 09 ± ±0 14 3*44 ± ± * Rat (5) 5-68 ± ± ± ± ±0-3 Mouse (6) 5-66 ± ± ± ± ±0-6 Ox (4) 7-58 ± ± ± V10 Sheep (4) 7-48 ± ± ± ± ± 1*3 Goat (1) Horse (4) 7.41 i ± ± ± Rabbit (10) 5.46 ± ±0-20 1*89 ± ± *20 ±0*7 Guinea-pig (6) 4-71 ± ± ± ±06 50 ± ±0*2 albumin peak was large, producing a high A/G ratio electrophoretically. The plasma lipoprotein concentration was low and lipid patterns stained faintly. Two regions of fat-staining could be identified, the first extending between the origin and the beta-globulin zone, whilst the second was a faintly stained area migrating with the alpha, globulins. This faster migrating lipoprotein could not be identified in all patterns due to its low concentration in some samples of rabbit plasma. Guinea-Pig.-The proteins separated into a small gamma globulin and fibrinogen peak with a small beta globulin and a large and welldefined alpha-globulin zone. The albumin peak was large and the A/G ratio high. Some samples of guinea-pig plasma showed a fast migrating component which moved in advance of the albumin spot. The concentration of lipids in the plasma was low and separation on filter paper poor. There was a small peak at the origin, followed by a lipid trail extending to the alpha-globulin area, where there was another small peak. Those samples of plasma which showed a fast migrating protein component also showed a corresponding area of lipid stain, indicating a lipoprotein of high electrophoretic mobility.

9 Animal Plasma Proteins and Lipids 135 Chemical Analyses.-The protein and lipid concentrations were determined in the plasmas, which were examined electrophoretically, and mean values for the different species are given in Table I. Cholesterol Distribution.-A qualitative estimate of the distribution of cholesterol along the electrophoretic patterns was made by spraying the papers with antimony pentachloride. Only in the human and dog, and in some samples of cat plasma, was the plasma cholesterol content sufficient to allow definite localization. In the human, the beta-lipoprotein area showed the presence of a large proportion of the plasma cholesterol, with only a relatively small amount present in the alpha, lipoprotein. In the dog and cat, however, the only areas of cholesterol localization were found in the fast migrating alpha-type lipoproteins that characterize these species. DIsCUSSION Different protein and lipoprotein fractions have been demonstrated in several animal species by moving boundary electrophoresis and ultracentrifugation [Moore, 1945; Deutsch and Goodloe, 1945; Lewis, Green and Page, 1952]. Our results, obtained by zone electrophoresis and chemical analysis, confirm that differences in the absolute amount and the relative distribution of both the protein and lipid constituents occur between animals of different dietary and digestive habits. In the plasma of all animals a variable amount of lipid stain is located near the origin in paper electrophoretic studies. This lipid peak, which is large during fat absorption and almost absent in the post-absorptive state, represents the concentration of particulate fat in the plasma [Swahn, 1953]. The true lipoprotein constituents do not change significantly during fat absorption [Gofman, Jones, Lindgren, Lyon, Elliott and Strisower, 1950; Page, Lewis and Plahl, 1953] and migrate on filter paper according to the electrophoretic mobility of their associated proteins. There appears, however, to be no simple correlation between dietary intake of fat and the distribution of lipids between the plasma proteins. In human plasma the beta globulins possess the highest affinity for lipid, whereas in the dog and cat, animals which have a diet similar to that of man, practically all the plasma lipids are bound by the alpha,-type globulins. As a group, the herbivora are characterized by low absolute amounts of plasma lipids and lipoprotein, which probably reflect the low dietary intake of fat. Cholesterol values are very low in all these animals, with the exception of the horse. The lipid patterns are similar between these species. There is an ill-defined zone of lipid migrating between the origin and the beta globulin, and a distinct and common lipoprotein which migrates just behind and with the slowest migrating albumin molecules. In the mouse particularly, and in some samples of rat and guinea-pig

10 136 Morris and Courtice plasma, there exists a characteristic lipoprotein with a rapid migration rate. This lipoprotein migrates in a very circumscribed area in advance of the main albumin zone. The lipid staining of this fraction is intense compared with the protein staining. The electrophoretic mobility suggests albumin as the protein constituent, and it appears that whatever lipids are involved in its constitution they are responsible for the increase in electrophoretic mobility. Robinson and French [1953], and Gordon, Boyle, Brown, Cherkes and Anfinsen [1953], have suggested a possible role for albumin in the mechanism of heparin clearing of lipaemic plasma. These authors suggest that albumin acts as a fatty acid acceptor for free fatty acids after the enzymatic hydrolysis of chylomicron neutral fat. Herbst and Hurley [1954] have recently reported the isolation of a prealbumin lipid component in samples of human serum following the intravenous injection of heparin, but were unable to demonstrate any corresponding protein component. The affinity of albumin for fatty acid anions is described by Ballou, Boyer and Luck [1945], who have demonstrated a significant increase in electrophoretic mobility of the albumin-fatty acid complex. The possibility that the pre-albumin lipoprotein seen constantly in the mouse and in some samples of guineapig and rat plasma might represent an albumin-fatty acid association is suggested. SUMMARY 1. The electrophoretic patterns and chemical composition of the plasma proteins and lipids of different animal species have been examined by zone electrophoresis on filter paper and by chemical analysis. 2. Significant differences have been found in the absolute amount and relative distribution of both the protein and lipid constituents of different animal plasmas. 3. Dog and cat plasma showed high levels of an alpha-type lipoprotein which contained most of the plasma cholesterol, whereas in human plasma cholesterol was mainly present in the large betalipoprotein fraction. 4. The herbivora in general had low levels of plasma lipoprotein and the concentration of alpha-type lipoprotein was relatively high. The horse had the highest concentrations of plasma lipids and lipoproteins of these animals. 5. The mouse constantly, and the rat and guinea-pig in some samples, showed the presence of a rapidly migrating fraction termed a pre-albumin lipoprotein. In the mouse this lipoprotein had a high proportion of lipid to protein and contained a large percentage of the plasma lipids.

11 Animal Plasma Proteins and Lipids 137 REFERENCES BALI OU, G. A., BOYER, R. D. and LUCK, J. M. (1945). J. biol. Chem. 159, 111. BLOOR, W. R. (1943) Biochemistry of the Fatty Acids. New York: Reinhold Publishing Corporation. CAMPBELL, W. R. and HANNA, M. I. (1937). J. biol. Chem. 119, 1. DEUTSCH, H. F. and GOODLOE, M. B. (1945). J. biol. Chem. 161, 1. FLYNN, F. V. and DE MAYO, P. (1951). Lancet, ii, 235. GORDON, R. S., BoNLE, E., BROWN, R. K., CHERKES, A. and ANFINSEN, C. B. (1953). Proc. Soc. exp. Biol. N.Y. 84, 168. GoFMAN, J. W., JONES, H. B., LINDGREN, F. T., LYON, T. P., ELLIOTT, H. A. and STRISOWER, B. (1950). Circulation, 2, 161. GURD, F. R. V., ONCLEY, J. L., EDSALL, J. T. and COHN, E. J. (1949). Di8c. Faraday Soc., No. 6. HERBST, F. S. and HURLEY, V. A. (1954). J. clin. Invest. 33, 907. KING, E. J. (1932). Biochem. J. 26, 292. KINGSLEY, G. R. and SCHAFFERT, R. R. (1949). J. biol. Chem. 180, 315. KUNKEL, H. G. and SLATER, R. J. (1952). J. cdin. Invest. 31, 677. LEWIS, L. A., GREEN, A. A. and PAGE, I. H. (1952). Amer. J. Phyaiol. 171, 391. MACHEB(EUF, M. (1929). Bull. Soc. Chim. biol. (Paris), 11, 268. MOORE, D. H. (1945). J. biol. Chem. 161, 21. ONCLEY, J. L. and GURD, F. R. V. (1953). In Blood Cells and Plasma Proteins. Edited by J. L. Tullis. New York: Academic Press, Inc. PAGE, I. H., LEWIS, L. A. and PLAHL, G. (1953). Circulation Research, 1, 87. ROBINSON, D. S. and FRENCH, J. E. (1953). Quart. J. exp. Physiol. 38, 233. ROSENBERG, I. V. (1952). Proc. Soc. exp. Biol. N.Y. 80, 751. STERNE, I. and SHAPIRO, B. (1953). J. clin. Path. 6, 158. SWAHN, B. (1952). Scand. J. clin. Lab. Invest. 4, 98. SWAHN, B. (1953). Scand. J. clin. Lab. Invest. 5, Supplementum 9.