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A comparison of the cellulose acetate electrophoretic serum lipoprotein-cholesterol profile of the adult male rat with other species
Comp. Biochem. Physiol. Vol. 83B, No. 1, pp. 37-44, 1986
0305-0491/86 $3.00 + 0.00 © 1986 Pergamon Press Ltd
Printed in Great Britain
A COMPARISON OF THE CELLULOSE ACETATE ELECTROPHORETIC SERUM LIPOPROTEINCHOLESTEROL PROFILE OF THE A D U L T MALE RAT WITH OTHER SPECIES JOSEPH R. DAVIS,* ANN R. DAVIS and I. BRUCE ROSENZWEIG Department of Pharmacology, Loyola University of Chicago Stritch School of Medicine, Maywood, IL 60153, USA (Tel: 312-531-3369)
(Received 15 May 1985) Abstract--1. A comparison was made of the serum lipoprotein-cholesterol profile, obtained by cellulose acetate electrophoresis coupled with an enzymatic stain for total cholesterol, of the adult male rat, mouse, rabbit, dog, monkey and human. 2. Four cholesterol-staining lipoprotein bands were detected in rat serum, while only three cholesterolstaining lipoprotein bands were present in the other species studied. 3. The apparently unique lipoprotein-cholesterol band in the rat was found to electrophoretically migrate in the prealbumin region of rat serum, has been named prealbumin lipoprotein-cholesterol (PAL-C) and was shown to be a high density lipoprotein (HDL). 4. Of the species studied those more susceptible to experimentally induced atherosclerosis had higher low density lipoprotein-cholesterol to HDL-cholesterol ratios compared to those species least susceptible to experimentally induced atherosclerosis.
INTRODUCTION High density lipoprotein-cholesterol ( H D L - C ) has been implicated as a protective factor against atherosclerotic disease (Gordon et al., 1977). Since the rat is known to have a very large proportionate amount of H D L - C (Chapman, 1980) and is also relatively resistant to the experimental induction of atherosclerotic disease (Filios et al., 1956; Wissler et al., 1954), this animal would appear to be an excellent model to study the relationship of high density lipoproteins to atherosclerosis. The coupling of an enzymatic determination of cholesterol (Allain et al., 1974) with cellulose acetate electrophoresis (Cobb and Sanders, 1978) has made possible a rapid and specific separation and analysis of human serum lipoprotein-cholesterol fractions, the ratios of which are currently being employed as risk factors for the development of human atherosclerosis. However, such a method has not been previously investigated with regard to the rat or other animal species besides the human. The present studies were therefore designed to compare the cellulose acetate electrophoretic serum lipoprotein-cholesterol profile of the rat with other animal species including the human. MATERIALS AND METHODS
Samples of rat (Rattus norvegicus) blood were obtained from the abdominal aorta of adult male Sprague-Dawley rats ranging from 60 to 90 days of age (Holtzman Co., Madison, WI, USA) under light ether anesthesia. Samples of rabbit (Oryctolagus cuniculus) blood were obtained from the ear vein of adult male New Zealand white rabbits *Correspondence to be addressed to: Joseph R. Davis, M.D., Ph.D., Professor of Pharmacology, Loyola University of Chicago Stritch School of Medicine, 2160 South First Ave., Maywood, IL 60153, USA. 37
(Langshaw Farms, Augusta, MI, USA). Samples of mouse (Mus musculus) blood were obtained from neck vessels following decapitation of adult male Cox Swiss mice (Lab Supply Co., Indianapolis, IN, USA). Samples of monkey blood were obtained from the femoral vein of phencyclidine-anesthetized adult male Rhesus monkeys (Macaca mulata) (Primate Imports, New York, NY, USA). Samples of dog blood were obtained from the femoral artery of ethrane and nitrous oxide-anesthetized adult male dogs (Canis familiaris) (Motsinger Kennels, St. Joseph, IL, USA). Samples of human (Homo sapiens) blood were obtained from the cubital vein of normal, healthy, adult male volunteers drawn while in a sitting position. Samples of blood were placed in an ice bath, allowed to clot for 1 hr and then centrifuged at 1000g for 15min at 4°C. The upper layer of serum was withdrawn and either placed in an ice bath for immediate analysis or frozen at -20°C for analysis done at a later time. Serum total cholesterol levels were measured using an enzymatic method (Allain et al., 1974). Electrophoresis was carried out according to the method of Cobb and Sanders (1978) on Titan III cellulose acetate plates (Helena Laboratories, Beaumont, TX, USA) that were soaked overnight in pH 8.8, 0.077 ionic strength tris-barbital buffer. Two microliters of serum were applied to the plate. The plate was electrophoresed at 180 V for 25 min. Ten min prior to the end of the electrophoresis period, a second plate which had soaked for at least 15 rain in 0.5 M, pH 6.7 phosphate buffer and blotted was then co,.'ered with enzymatic cholesterol-staining reagent (Helena Laboratories). The electrophoresed sample plate was layered carefully on top of the reagent plate and excess reagent and air bubbles removed, making a sandwich of the two plates. The sandwiched plates were placed between two preheated development weights and incubated on top of a Helena Laboratories microevaporator hood for 25 min at 37°C, following which the two plates were separated and the sample plate dried and examined under transmitted light for orange-staining lipoprotein-cholesterol bands. The orangestaining lipoprotein-cholesterol plate was densitometrically scanned at 500 nm in a Beckman DU-8 spectrophotometer.
38
JOSEPH R. DAVISet al.
Half of the original sample plate containing duplicate samples of serum was stained for protein with Ponceau S and densitometrically scanned at 525 nm (Briere and Mull, 1964). Ultracentrifugal isolation of the rat serum HDL fraction was performed according to the method of Havel et al. (1955). Following the removal of the density less than 1.063 g/ml fraction from 9 ml of serum by centrifugation using a type 40 rotor in a Beckman Model L ultracentrifuge at 40,000 rpm for 24 hr at 15°C, the infranate was adjusted to a density of 1.210 g/ml by adding a concentrated salt solution of NaC1 and KBr (density 1.346 g/ml). The sample was mixed and centrifuged at 40,000 rpm for 24 hr at 15°C. the top 1.0ml of the supernate representing the HDL fraction (density 1.063-1.210g/ml) was removed, washed with a density 1.210 g/ml salt solution and centrifuged at 40,000rpm for 24hr. The final washed 1.063-1.210g/ml
ADULT
density HDL fraction was dialyzed overnight at 4°C against 0.15 M NaC1 solution containing 0.05% EDTA and then electrophoresed on a cellulose acetate plate as described above. Manganese beparin precipitation of rat serum lower density lipoproteins was performed using a modification of the Lipid Research Clinics Program Method (Manual of Laboratory Operations, 1974). A 0.I ml aliquot of HDL reagent containing 1012 mmol/1 manganese chloride and 2000 U/ml heparin (Worthington Diagnostics, Freehold, N J, USA) was added to 1.0 ml of serum. The sample was vortexed 5 sec, placed in an ice bath for 30 min and then centrifuged at 1000g for 30 min at 4°C. the supernate containing the HDL-cholesterol was removed, dialyzed overnight at 4°C against 0.15M NaCI containing 0.05% EDTA and then electrophoresed on a cellulose acetate plate as described above.
MALE
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Comparison of lipoprotein-cholesterol profiles
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Table 1. Serum lipoprotein~holesterol values of normal, non-fasting animals
n
Total cholesterol (mg/dl) Mean _+SD
PAL-C (mg/dl) Mean_+ SD
cqL-C (rag/d1) Mean_+ SD
~%L C (mg/dl) Mean_+ SD
15 _+4
33 _+ 7
3+ 1
flL~2 (mg/dl) Mean + SD
LDL-C* HDL-C
Total C* HDL-C
8+ 2
0.17
1.23
Adult male
Rattus norvegicus
15
59 + 11
6
127+20
0
109+15
7+3
11+5
0.10
1.16
10
34 + 8
0
20 _+6
3+ 2
l1+ 3
0.55
1.68
3
139_+ 15
0
116_+ 11
12_+2.
11 _+3
0.09
1.20
3
147 _+ 25
0
89 + 30
12 _+ 5
46 _+ 5
0.52
1.65
13
183 + 47
0
44 _+ 9
12 _+6
127 _+46
3.05
4.33
Adult male
Musmusculus Adult male
Oryctolagus cuniculus Adult male
Canisfamiliaris Adult male
Macaca mulatta Adult male
Homo sapiens (21-39 yr)
*To calculate the comparative ratios (2), LDL C is assumed to be equivalent to flL-C and H D L - C is assumed to be equivalent to PAL42 + :qL C.
RESULTS
Figure l presents a comparison of the cellulose acetate electrophoretic migration of serum proteins of the adult male rat stained for both total cholesterol and for protein. Four orange-colored cholesterolstaining lipoprotein bands were detected in the serum of adult male rats and have been named according to their plasma protein-staining electrophoretic mobilities. Beta lipoprotein-cholesterol, abbreviated as ilL-C, had ill-globulin mobility corresponding to low density lipoprotein (LDL) and accounted for an average of 13.6% of the total plasma lipoprotein-cholesterol. Alpha2 lipoproteincholesterol, abbreviated as ~2L-C, had ~2-globulin mobility corresponding to very low density lipoprotein (VLDL) and accounted for 5.1% of the total plasma lipoprotein-cholesterol. Alpha~ lipoproteincholesterol, abbreviated as CtlL~C, had cq-globulin mobility corresponding to high density lipoprotein (HDL) and accounted for 55.9% of the total plasma lipoprotein-cholesterol. A fourth band was found to migrate faster than rat albumin with prealbumin mobility accounting for the remaining 25.4% of the total plasma lipoprotein-cholesterol and has been presently named as prealbumin lipoprotein cholesterol and abbreviated as PAL-C. Overnightfasting of adult male rats did not affect the relative percentages of the presently obtained lipoproteincholesterol profile. Figure 2 presents the comparative distribution of plasma lipoprotein-cholesterol fractions separated by cellulose acetate electrophoresis in normal adult male rat, mouse, rabbit, dog, monkey and human serum samples. P A L - C found in rat serum was not present in serum samples of the adult mouse, rabbit, dog, monkey or human. These data suggest that P A L - C may be a new plasma lipoprotein-cholesterol fraction with a cellulose acetate electrophoretic mobility faster than albumin that may be unique to the rat. Serum lipoproein-cholesterol values in terms of mg/dl of the various normal adult, non-fasting animals studied by cellulose acetate electrophoresis are
given in Table 1. The concentration of P A L - C and cqL-C in adult male rat serum was found to be 15 _+ 4 and 33 _+ 7 mg/dl, respectively. The sum of P A L - C and ~IL-C of the adult male rat was found to be 81.4% of the total serum cholesterol concentration which appears to agree with previous reports in the literature for the total amount of rat serum high density lipoprotein-cholesterol as determined by ultracentrifugal separation of lipoproteins (Havel et al., 1955; Narayan, 1971). One of the currently-used coronary artery disease risk ratios obtained by dividing the LDL-cholesterol (LDL-C) concentration by the HDL-cholesterol (HDL-C) concentration (Gordon et al., 1977) was found to be lowest for those species least responsive to diet-induced atherogenesis (rat, mouse and dog) (Beher et al., 1963; Mahley et al., 1974) and highest in those species (rabbit, monkey and human) most prone to diet-induced atherogenesis (Camejo et al., 1973; Kramsch et al., 1981; Mahley, 1978). In addition, the adult male human was also found to differ markedly from the experimental animals studied with regard to another currently-employed coronary artery disease risk ratio (Gordon et al., 1977) having a serum total cholesterol to HDL-cholesterol ratio almost three times as great as the other species. Figure 3 characterizes P A L - C and cqL-C separated by cellulose acetate electrophoresis as rat serum high density lipoprotein-cholesterol fractions employing ultracentrifugation (Havel et al., 1955). Only two cholesterol-staining bands were found to occur in the high density fraction (1.063-1.210g/ml) of normal adult rat serum separated by ultracentrifugation. These two bands had cellulose acetate electrophoretic mobilities corresponding to P A L - C and cqL-C, indicating that both belong to the HDL-cholesterol fraction of rat plasma. In addition, Fig. 4 shows that both PAL and cqL of the adult rat were also found in the high density lipoprotein supernate using a polyanionic precipitation method (Manual of Laboratory Operations, 1974). The manganese heparin supernate was dialyzed prior to cellulose acetate
Comparison of lipoprotein-cholesterol profiles
41
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DISTANCE FROM POINT OF SAMPLE APPLICATION (ram) Fig. 3. Cellulose acetate electrophoresis of the high density fraction (1.063-1.210 g/ml) of adult male rat serum obtained by ultracentrifugation. A sample of the corresponding whole serum was stored at -20°C during the time required to isolate the high density fraction, and was subsequently electrophoresed on the same plate. electrophoresis in order to remove the manganese, since manganese interferes with the enzymatic cholesterol reagent (Steele et al., 1976). Two cholesterolstaining bands occurred in the 1000g manganese heparin high density lipoprotein supernate, with electrophoretic mobilities corresponding to P A L ~ and alL-C.
DISCUSSION Recently, a cellulose acetate electrophoretic technique combining an enzymatic staining procedure for total cholesterol (Allain et al., 1974) has been developed for determining human serum
lipoprotein-cholesterol concentrations currently employed as risk factors for human atherosclerotic disease (Cobb and Sanders, 1978). However, this technique has not previously been used to identify rat or other animal species except for human serum lipoprotein-cholesterol fractions. The present data employing cellulose acetate electrophoresis combined with an enzymatic cholesterol stain clearly demonstrates for the first time that adult male rat serum contains a rapidly-moving high density lipoprotein-cholesterol fraction with an electrophoretic mobility equivalent to prealbumin and which has been designated as prealbumin lipoprotein-cholesterol or PAL-C. PAL-C was not
42
JOSEPH R. DAVISet al. ADULT ELECTROPHORESIS 0.077
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Fig. 4. Cellulose acetate electrophoresis of the dialyzed supernate obtained after manganese heparin precipitation of adult male rat serum. A sample of the corresponding whole serum was stored at - 20°C during the time required to dialyze the manganese heparin supernate against 0.15 M sodium chloride solution containing 0.05% EDTA at 4°C, and was subsequently electrophoresed on the same plate. found in serum samples of the adult mouse, rabbit, dog, monkey or human, suggesting that it may be unique to the rat. Previous electrophoretic studies of rat serum apparently have not demonstrated prealbumin lipoprotein due to either differences in the electrophoretic medium or due to the semiquantitative nature of the "total" lipid stains employed. Accurate quantitation of electrophoretically-separated plasma lipoproteins with non-specific "total" lipid stains has been difficult, because of each lipoprotein's differing affinities for such stains (Fletcher and Styliou, 1970). Such commonly used lipid stains as Sudan black B, preferentially stain unsaturated sterol esters and un-
saturated triglycerides, whereas unesterified cholesterol and phospholipids are stained negligibly or not at all (Schjeide et al., 1963). Moreover, the various cholesterol esters and triglycerides have different staining affinities for Sudan black B, dependent upon their particular fatty acids. Oil red O, which is another commonly used "total" lipid stain, stains cholesterol esters more intensely than Sudan black B, but its staining is also dependent on the cholesterol ester's degree of fatty acid saturation. In addition, Oil red O also stains triglycerides as well as albumin, but does not stain phospholipids or unesterified cholesterol. Agarose gel electrophoresis of rat serum HDL following ultracentrifugal separation demonstrated a
Comparison of lipoprotein-cholesterol profiles
43
single lipid-staining band whose migration appeared in this laboratory to establish whether a relationship to include the prealbumin region (Koga et al., 1969). between ~tlL and PAL exists in rat cholesterol metabAgarose gel electrophoresis of rat serum combined olism. with a Sudan black B stain also appeared to suggest a prealbumin lipoprotein band; however in this sysREFERENCES tem lipid-stained bands migrated consistently farther than their corresponding amido black protein-stained Allain C. C., Poon L. S., Chan C. S. G., Richmond W. and Fu P. C. (1974) Enzymatic determination of total serum bands (Johansson and Karlsson, 1976). In addition, cholesterol. Clin. Chem. 20, 470-473. agarose-starch gel electrophoresis of rat serum using Beher W. T., Baker G. D. and Penney D. G. (1963) a combined Oil red O and fat 7B "total" lipid stain Comparative study of the effects of bile acids and cholesalso appeared to suggest bands which had preterol on cholesterol metabolism in the mouse, rat, hamster albumin, alphas, prebeta and beta electrophoretic and guinea pig. J. Nutr. 79, 523-530. mobility (Chalvardjian, 1971). The prealbumin and Briere R. O. and Mull J. D. (1964) Electrophoresis of serum protein with cellulose acetate: a method for quantitation. alpha~ bands appeared to correspond to HDL, since Am. J. clin. Path. 42, 547-55I. after ultracentrifugal fractionation of the serum, these two bands had a density range greater than Camejo G., Bosch V., Arreaza C. and Mendez H. C. (1973) Early changes in plasma lipoprotein structure and bio!.063 g/ml, while the prcbeta band corresponded to synthesis in cholesterol-fed rabbits. J. Lipid Res. 14, VLDL and the beta band corresponded to LDL. 61-68. However, the prealbumin lipid-staining band was Chalvardjian A. (1971) Agarose-starch gel electrophoresis distinguished from albumin only by the difference in of rat serum lipoproteins. J. Lipid Res. 12, 265-269. albumin's staining color and no attempt was made to Chapman M. J. (1980) Animal lipoproteins: chemistry, structure and comparative aspects. J. Lipid Res. 21, determine the actual cholesterol content of the 789-853. prealbumin-migrating lipoprotein band visualized by the "total" lipid stain. Moreover, a side-by-side elec- Cobb S. A. and Sanders J. L. (1978) Enzymic determination of cholesterol in serum lipoproteins separated by electrotrophoretic comparison of total lipid-stained and phoresis. Clin. Chem. 24, Ill6-1120. protein-stained serum was not performed using the Fillios L. C., Andrus S. B., Mann G. V. and Stare F. J. agarose-starch gel electrophoretic technique. On the (1956) Experimental production of gross atherosclerosis other hand, the present studies employing cellulose in the rat. J. exp. Med. 104, 539-554. acetate electrophoresis combined with an enzymatic Fletcher M. J. and Styliou M. (1970) A simple method for cholesterol stain allows for an easy distinction beseparating serum lipoproteins by electrophoresis on cellulose acetate. Clin. Chem. 16, 362-365. tween prealbumin lipoprotein and albumin, as well as a more accurate measure of rat serum Gordon T., Castelli W., Hjortland M., Kannel W. and Dawber T. (1977) High density lipoprotein as a protective lipoprotein-cholesterol concentrations utilizing only factor against coronary heart disease, The Framingham a 2/~1 sample of serum which has the advantage of study. Am. J. Med. 62, 707-714. permitting multiple sampling from the same animal. Havel R. J., Eder H. A. and Bragdon J. H. (1955) The Serum lipoprotein-cholesterol ratios for various distribution and chemical composition of ultraanimal species have been obtained employing cellucentrifugally separated lipoproteins in human serum. J. lose acetate electrophoretic separaton of lipoproteins clin. Incest. 34, 1345-1353. coupled with an enzymatic cholesterol stain. The Johansson M. B. and Karlsson B. W. (1976) Lipoproteins in serum of rat, mouse, gerbil, rabbit, pig and man studied L D L - C / H D L - C ratios were presently found to be by electrophoretical and immunological methods. Comp. markedly higher in the rabbit, monkey and human, Biochem. Physiol. 54B, 495-500. species known to be more prone to experimentallyinduced atherosclerotic lesions (Beher et al., 1963; Koga S., Horwitz D. L. and Scanu A. M. (1969) Isolation and properties of lipoproteins from normal rat serum. J. Mahley et al., 1974) than the L D L - C / H D L - C ratios Lipid Res. 10, 577-588. of the rat, mouse and dog, which are species less Kramsch D. M., Aspen A. J. and Rozler L. J. (1981) prone to induced atherosclerotic lesions (Wissler et Atherosclerosis: prevention by agents not affectingabnoraL, 1954; Fillios et al., 1956; Camejo et al., 1973; mal levels of blood lipids. Science 213, 1511-1512. Mahley, 1978; Kramsch et al., 1981). The total Mahley R. W. (1978) Disturbances in Lipid and Lipoprotein cholesterol to H D L - C ratio was also found to be Metabolism, pp. 181-197. Williams and Wilkins, Baltimore, MD. higher in those species most prone to induced atherosclerotic lesions, although the species differences be- Mahley R. W., Weisgraber K. H. and Innerarity T. (1974) Canine lipoproteins and atherosclerosis II. Charactertween the total cholesterol/HDL-C ratios appear to ization of the plasma lipoproteins associated with atherobe less marked than the species differences between genic and nonatherogenic hyperlipidemia. Circulation the L D L ~ 2 / H D L - C ratios. Human epidemiological Res. 35, 722-733. evidence has shown that the L D L - C / H D L - C ratio is Manual of Laboratory Operations (1974) Lipid Research positively correlated to the risk of developing atheroClinics Programs. DHEW Publication No. (NIH) 75-628. sclerotic disease (Chapman, 1980), therefore it is Narayan K. A. (197I) Lowered serum concentration of high possible that a high L D L - C / H D L - C ratio may be an density lipoproteins in cholesterol-fed rats. Atherosclerosis 13, 205-215. important indicator of species susceptability to the experimental induction of atherosclerotic lesions. In Oschry Y. and Eisenberg S. (1982) Rat plasma lipoproteins: reevaluation of a lipoprotein system in an animal devoid addition, since it has been reported that cholesterol of cholesterol ester transfer activity. J. Lipid Res. 23, ester transfer does not occur between rat plasma 1099-1106. lipoproteins (Oschry and Eisenberg, 1982) the possi- Schjeide O. A., Rivin A. U. and Yoshino J. (1963) Uptake bility exists that rat prealbumin lipoprotein (PAL) of lipid stains by lipids and serum lipoproteins. Am. J. may have a role in the clearance of cholesterol from clin, Path. 39, 329 341. rat plasma and experiments are currently in progress Steele B. W., Koehler D. F., Azar M. M., Blaszkowski T.
44
JOSEPH R. DAVIS et al. P., Kuba K. and Dempsey M. E. (1976) Enzymatic determination of cholesterol in high-density-lipoprotein fractions prepared by a precipitation technique. Clin. Chem. 22, 98-101.
Wissler R. W., Eilert M. L., Schroeder M. A. and Cohen L. (1954) Production of lipomatous and atheromatous arterial lesions in the albino rat. A. M. A. Archs Path. 57, 333-351.
0305-0491/86 $3.00 + 0.00 © 1986 Pergamon Press Ltd
Printed in Great Britain
A COMPARISON OF THE CELLULOSE ACETATE ELECTROPHORETIC SERUM LIPOPROTEINCHOLESTEROL PROFILE OF THE A D U L T MALE RAT WITH OTHER SPECIES JOSEPH R. DAVIS,* ANN R. DAVIS and I. BRUCE ROSENZWEIG Department of Pharmacology, Loyola University of Chicago Stritch School of Medicine, Maywood, IL 60153, USA (Tel: 312-531-3369)
(Received 15 May 1985) Abstract--1. A comparison was made of the serum lipoprotein-cholesterol profile, obtained by cellulose acetate electrophoresis coupled with an enzymatic stain for total cholesterol, of the adult male rat, mouse, rabbit, dog, monkey and human. 2. Four cholesterol-staining lipoprotein bands were detected in rat serum, while only three cholesterolstaining lipoprotein bands were present in the other species studied. 3. The apparently unique lipoprotein-cholesterol band in the rat was found to electrophoretically migrate in the prealbumin region of rat serum, has been named prealbumin lipoprotein-cholesterol (PAL-C) and was shown to be a high density lipoprotein (HDL). 4. Of the species studied those more susceptible to experimentally induced atherosclerosis had higher low density lipoprotein-cholesterol to HDL-cholesterol ratios compared to those species least susceptible to experimentally induced atherosclerosis.
INTRODUCTION High density lipoprotein-cholesterol ( H D L - C ) has been implicated as a protective factor against atherosclerotic disease (Gordon et al., 1977). Since the rat is known to have a very large proportionate amount of H D L - C (Chapman, 1980) and is also relatively resistant to the experimental induction of atherosclerotic disease (Filios et al., 1956; Wissler et al., 1954), this animal would appear to be an excellent model to study the relationship of high density lipoproteins to atherosclerosis. The coupling of an enzymatic determination of cholesterol (Allain et al., 1974) with cellulose acetate electrophoresis (Cobb and Sanders, 1978) has made possible a rapid and specific separation and analysis of human serum lipoprotein-cholesterol fractions, the ratios of which are currently being employed as risk factors for the development of human atherosclerosis. However, such a method has not been previously investigated with regard to the rat or other animal species besides the human. The present studies were therefore designed to compare the cellulose acetate electrophoretic serum lipoprotein-cholesterol profile of the rat with other animal species including the human. MATERIALS AND METHODS
Samples of rat (Rattus norvegicus) blood were obtained from the abdominal aorta of adult male Sprague-Dawley rats ranging from 60 to 90 days of age (Holtzman Co., Madison, WI, USA) under light ether anesthesia. Samples of rabbit (Oryctolagus cuniculus) blood were obtained from the ear vein of adult male New Zealand white rabbits *Correspondence to be addressed to: Joseph R. Davis, M.D., Ph.D., Professor of Pharmacology, Loyola University of Chicago Stritch School of Medicine, 2160 South First Ave., Maywood, IL 60153, USA. 37
(Langshaw Farms, Augusta, MI, USA). Samples of mouse (Mus musculus) blood were obtained from neck vessels following decapitation of adult male Cox Swiss mice (Lab Supply Co., Indianapolis, IN, USA). Samples of monkey blood were obtained from the femoral vein of phencyclidine-anesthetized adult male Rhesus monkeys (Macaca mulata) (Primate Imports, New York, NY, USA). Samples of dog blood were obtained from the femoral artery of ethrane and nitrous oxide-anesthetized adult male dogs (Canis familiaris) (Motsinger Kennels, St. Joseph, IL, USA). Samples of human (Homo sapiens) blood were obtained from the cubital vein of normal, healthy, adult male volunteers drawn while in a sitting position. Samples of blood were placed in an ice bath, allowed to clot for 1 hr and then centrifuged at 1000g for 15min at 4°C. The upper layer of serum was withdrawn and either placed in an ice bath for immediate analysis or frozen at -20°C for analysis done at a later time. Serum total cholesterol levels were measured using an enzymatic method (Allain et al., 1974). Electrophoresis was carried out according to the method of Cobb and Sanders (1978) on Titan III cellulose acetate plates (Helena Laboratories, Beaumont, TX, USA) that were soaked overnight in pH 8.8, 0.077 ionic strength tris-barbital buffer. Two microliters of serum were applied to the plate. The plate was electrophoresed at 180 V for 25 min. Ten min prior to the end of the electrophoresis period, a second plate which had soaked for at least 15 rain in 0.5 M, pH 6.7 phosphate buffer and blotted was then co,.'ered with enzymatic cholesterol-staining reagent (Helena Laboratories). The electrophoresed sample plate was layered carefully on top of the reagent plate and excess reagent and air bubbles removed, making a sandwich of the two plates. The sandwiched plates were placed between two preheated development weights and incubated on top of a Helena Laboratories microevaporator hood for 25 min at 37°C, following which the two plates were separated and the sample plate dried and examined under transmitted light for orange-staining lipoprotein-cholesterol bands. The orangestaining lipoprotein-cholesterol plate was densitometrically scanned at 500 nm in a Beckman DU-8 spectrophotometer.
38
JOSEPH R. DAVISet al.
Half of the original sample plate containing duplicate samples of serum was stained for protein with Ponceau S and densitometrically scanned at 525 nm (Briere and Mull, 1964). Ultracentrifugal isolation of the rat serum HDL fraction was performed according to the method of Havel et al. (1955). Following the removal of the density less than 1.063 g/ml fraction from 9 ml of serum by centrifugation using a type 40 rotor in a Beckman Model L ultracentrifuge at 40,000 rpm for 24 hr at 15°C, the infranate was adjusted to a density of 1.210 g/ml by adding a concentrated salt solution of NaC1 and KBr (density 1.346 g/ml). The sample was mixed and centrifuged at 40,000 rpm for 24 hr at 15°C. the top 1.0ml of the supernate representing the HDL fraction (density 1.063-1.210g/ml) was removed, washed with a density 1.210 g/ml salt solution and centrifuged at 40,000rpm for 24hr. The final washed 1.063-1.210g/ml
ADULT
density HDL fraction was dialyzed overnight at 4°C against 0.15 M NaC1 solution containing 0.05% EDTA and then electrophoresed on a cellulose acetate plate as described above. Manganese beparin precipitation of rat serum lower density lipoproteins was performed using a modification of the Lipid Research Clinics Program Method (Manual of Laboratory Operations, 1974). A 0.I ml aliquot of HDL reagent containing 1012 mmol/1 manganese chloride and 2000 U/ml heparin (Worthington Diagnostics, Freehold, N J, USA) was added to 1.0 ml of serum. The sample was vortexed 5 sec, placed in an ice bath for 30 min and then centrifuged at 1000g for 30 min at 4°C. the supernate containing the HDL-cholesterol was removed, dialyzed overnight at 4°C against 0.15M NaCI containing 0.05% EDTA and then electrophoresed on a cellulose acetate plate as described above.
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Comparison of lipoprotein-cholesterol profiles
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Table 1. Serum lipoprotein~holesterol values of normal, non-fasting animals
n
Total cholesterol (mg/dl) Mean _+SD
PAL-C (mg/dl) Mean_+ SD
cqL-C (rag/d1) Mean_+ SD
~%L C (mg/dl) Mean_+ SD
15 _+4
33 _+ 7
3+ 1
flL~2 (mg/dl) Mean + SD
LDL-C* HDL-C
Total C* HDL-C
8+ 2
0.17
1.23
Adult male
Rattus norvegicus
15
59 + 11
6
127+20
0
109+15
7+3
11+5
0.10
1.16
10
34 + 8
0
20 _+6
3+ 2
l1+ 3
0.55
1.68
3
139_+ 15
0
116_+ 11
12_+2.
11 _+3
0.09
1.20
3
147 _+ 25
0
89 + 30
12 _+ 5
46 _+ 5
0.52
1.65
13
183 + 47
0
44 _+ 9
12 _+6
127 _+46
3.05
4.33
Adult male
Musmusculus Adult male
Oryctolagus cuniculus Adult male
Canisfamiliaris Adult male
Macaca mulatta Adult male
Homo sapiens (21-39 yr)
*To calculate the comparative ratios (2), LDL C is assumed to be equivalent to flL-C and H D L - C is assumed to be equivalent to PAL42 + :qL C.
RESULTS
Figure l presents a comparison of the cellulose acetate electrophoretic migration of serum proteins of the adult male rat stained for both total cholesterol and for protein. Four orange-colored cholesterolstaining lipoprotein bands were detected in the serum of adult male rats and have been named according to their plasma protein-staining electrophoretic mobilities. Beta lipoprotein-cholesterol, abbreviated as ilL-C, had ill-globulin mobility corresponding to low density lipoprotein (LDL) and accounted for an average of 13.6% of the total plasma lipoprotein-cholesterol. Alpha2 lipoproteincholesterol, abbreviated as ~2L-C, had ~2-globulin mobility corresponding to very low density lipoprotein (VLDL) and accounted for 5.1% of the total plasma lipoprotein-cholesterol. Alpha~ lipoproteincholesterol, abbreviated as CtlL~C, had cq-globulin mobility corresponding to high density lipoprotein (HDL) and accounted for 55.9% of the total plasma lipoprotein-cholesterol. A fourth band was found to migrate faster than rat albumin with prealbumin mobility accounting for the remaining 25.4% of the total plasma lipoprotein-cholesterol and has been presently named as prealbumin lipoprotein cholesterol and abbreviated as PAL-C. Overnightfasting of adult male rats did not affect the relative percentages of the presently obtained lipoproteincholesterol profile. Figure 2 presents the comparative distribution of plasma lipoprotein-cholesterol fractions separated by cellulose acetate electrophoresis in normal adult male rat, mouse, rabbit, dog, monkey and human serum samples. P A L - C found in rat serum was not present in serum samples of the adult mouse, rabbit, dog, monkey or human. These data suggest that P A L - C may be a new plasma lipoprotein-cholesterol fraction with a cellulose acetate electrophoretic mobility faster than albumin that may be unique to the rat. Serum lipoproein-cholesterol values in terms of mg/dl of the various normal adult, non-fasting animals studied by cellulose acetate electrophoresis are
given in Table 1. The concentration of P A L - C and cqL-C in adult male rat serum was found to be 15 _+ 4 and 33 _+ 7 mg/dl, respectively. The sum of P A L - C and ~IL-C of the adult male rat was found to be 81.4% of the total serum cholesterol concentration which appears to agree with previous reports in the literature for the total amount of rat serum high density lipoprotein-cholesterol as determined by ultracentrifugal separation of lipoproteins (Havel et al., 1955; Narayan, 1971). One of the currently-used coronary artery disease risk ratios obtained by dividing the LDL-cholesterol (LDL-C) concentration by the HDL-cholesterol (HDL-C) concentration (Gordon et al., 1977) was found to be lowest for those species least responsive to diet-induced atherogenesis (rat, mouse and dog) (Beher et al., 1963; Mahley et al., 1974) and highest in those species (rabbit, monkey and human) most prone to diet-induced atherogenesis (Camejo et al., 1973; Kramsch et al., 1981; Mahley, 1978). In addition, the adult male human was also found to differ markedly from the experimental animals studied with regard to another currently-employed coronary artery disease risk ratio (Gordon et al., 1977) having a serum total cholesterol to HDL-cholesterol ratio almost three times as great as the other species. Figure 3 characterizes P A L - C and cqL-C separated by cellulose acetate electrophoresis as rat serum high density lipoprotein-cholesterol fractions employing ultracentrifugation (Havel et al., 1955). Only two cholesterol-staining bands were found to occur in the high density fraction (1.063-1.210g/ml) of normal adult rat serum separated by ultracentrifugation. These two bands had cellulose acetate electrophoretic mobilities corresponding to P A L - C and cqL-C, indicating that both belong to the HDL-cholesterol fraction of rat plasma. In addition, Fig. 4 shows that both PAL and cqL of the adult rat were also found in the high density lipoprotein supernate using a polyanionic precipitation method (Manual of Laboratory Operations, 1974). The manganese heparin supernate was dialyzed prior to cellulose acetate
Comparison of lipoprotein-cholesterol profiles
41
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DISTANCE FROM POINT OF SAMPLE APPLICATION (ram) Fig. 3. Cellulose acetate electrophoresis of the high density fraction (1.063-1.210 g/ml) of adult male rat serum obtained by ultracentrifugation. A sample of the corresponding whole serum was stored at -20°C during the time required to isolate the high density fraction, and was subsequently electrophoresed on the same plate. electrophoresis in order to remove the manganese, since manganese interferes with the enzymatic cholesterol reagent (Steele et al., 1976). Two cholesterolstaining bands occurred in the 1000g manganese heparin high density lipoprotein supernate, with electrophoretic mobilities corresponding to P A L ~ and alL-C.
DISCUSSION Recently, a cellulose acetate electrophoretic technique combining an enzymatic staining procedure for total cholesterol (Allain et al., 1974) has been developed for determining human serum
lipoprotein-cholesterol concentrations currently employed as risk factors for human atherosclerotic disease (Cobb and Sanders, 1978). However, this technique has not previously been used to identify rat or other animal species except for human serum lipoprotein-cholesterol fractions. The present data employing cellulose acetate electrophoresis combined with an enzymatic cholesterol stain clearly demonstrates for the first time that adult male rat serum contains a rapidly-moving high density lipoprotein-cholesterol fraction with an electrophoretic mobility equivalent to prealbumin and which has been designated as prealbumin lipoprotein-cholesterol or PAL-C. PAL-C was not
42
JOSEPH R. DAVISet al. ADULT ELECTROPHORESIS 0.077
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RAT
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Fig. 4. Cellulose acetate electrophoresis of the dialyzed supernate obtained after manganese heparin precipitation of adult male rat serum. A sample of the corresponding whole serum was stored at - 20°C during the time required to dialyze the manganese heparin supernate against 0.15 M sodium chloride solution containing 0.05% EDTA at 4°C, and was subsequently electrophoresed on the same plate. found in serum samples of the adult mouse, rabbit, dog, monkey or human, suggesting that it may be unique to the rat. Previous electrophoretic studies of rat serum apparently have not demonstrated prealbumin lipoprotein due to either differences in the electrophoretic medium or due to the semiquantitative nature of the "total" lipid stains employed. Accurate quantitation of electrophoretically-separated plasma lipoproteins with non-specific "total" lipid stains has been difficult, because of each lipoprotein's differing affinities for such stains (Fletcher and Styliou, 1970). Such commonly used lipid stains as Sudan black B, preferentially stain unsaturated sterol esters and un-
saturated triglycerides, whereas unesterified cholesterol and phospholipids are stained negligibly or not at all (Schjeide et al., 1963). Moreover, the various cholesterol esters and triglycerides have different staining affinities for Sudan black B, dependent upon their particular fatty acids. Oil red O, which is another commonly used "total" lipid stain, stains cholesterol esters more intensely than Sudan black B, but its staining is also dependent on the cholesterol ester's degree of fatty acid saturation. In addition, Oil red O also stains triglycerides as well as albumin, but does not stain phospholipids or unesterified cholesterol. Agarose gel electrophoresis of rat serum HDL following ultracentrifugal separation demonstrated a
Comparison of lipoprotein-cholesterol profiles
43
single lipid-staining band whose migration appeared in this laboratory to establish whether a relationship to include the prealbumin region (Koga et al., 1969). between ~tlL and PAL exists in rat cholesterol metabAgarose gel electrophoresis of rat serum combined olism. with a Sudan black B stain also appeared to suggest a prealbumin lipoprotein band; however in this sysREFERENCES tem lipid-stained bands migrated consistently farther than their corresponding amido black protein-stained Allain C. C., Poon L. S., Chan C. S. G., Richmond W. and Fu P. C. (1974) Enzymatic determination of total serum bands (Johansson and Karlsson, 1976). In addition, cholesterol. Clin. Chem. 20, 470-473. agarose-starch gel electrophoresis of rat serum using Beher W. T., Baker G. D. and Penney D. G. (1963) a combined Oil red O and fat 7B "total" lipid stain Comparative study of the effects of bile acids and cholesalso appeared to suggest bands which had preterol on cholesterol metabolism in the mouse, rat, hamster albumin, alphas, prebeta and beta electrophoretic and guinea pig. J. Nutr. 79, 523-530. mobility (Chalvardjian, 1971). The prealbumin and Briere R. O. and Mull J. D. (1964) Electrophoresis of serum protein with cellulose acetate: a method for quantitation. alpha~ bands appeared to correspond to HDL, since Am. J. clin. Path. 42, 547-55I. after ultracentrifugal fractionation of the serum, these two bands had a density range greater than Camejo G., Bosch V., Arreaza C. and Mendez H. C. (1973) Early changes in plasma lipoprotein structure and bio!.063 g/ml, while the prcbeta band corresponded to synthesis in cholesterol-fed rabbits. J. Lipid Res. 14, VLDL and the beta band corresponded to LDL. 61-68. However, the prealbumin lipid-staining band was Chalvardjian A. (1971) Agarose-starch gel electrophoresis distinguished from albumin only by the difference in of rat serum lipoproteins. J. Lipid Res. 12, 265-269. albumin's staining color and no attempt was made to Chapman M. J. (1980) Animal lipoproteins: chemistry, structure and comparative aspects. J. Lipid Res. 21, determine the actual cholesterol content of the 789-853. prealbumin-migrating lipoprotein band visualized by the "total" lipid stain. Moreover, a side-by-side elec- Cobb S. A. and Sanders J. L. (1978) Enzymic determination of cholesterol in serum lipoproteins separated by electrotrophoretic comparison of total lipid-stained and phoresis. Clin. Chem. 24, Ill6-1120. protein-stained serum was not performed using the Fillios L. C., Andrus S. B., Mann G. V. and Stare F. J. agarose-starch gel electrophoretic technique. On the (1956) Experimental production of gross atherosclerosis other hand, the present studies employing cellulose in the rat. J. exp. Med. 104, 539-554. acetate electrophoresis combined with an enzymatic Fletcher M. J. and Styliou M. (1970) A simple method for cholesterol stain allows for an easy distinction beseparating serum lipoproteins by electrophoresis on cellulose acetate. Clin. Chem. 16, 362-365. tween prealbumin lipoprotein and albumin, as well as a more accurate measure of rat serum Gordon T., Castelli W., Hjortland M., Kannel W. and Dawber T. (1977) High density lipoprotein as a protective lipoprotein-cholesterol concentrations utilizing only factor against coronary heart disease, The Framingham a 2/~1 sample of serum which has the advantage of study. Am. J. Med. 62, 707-714. permitting multiple sampling from the same animal. Havel R. J., Eder H. A. and Bragdon J. H. (1955) The Serum lipoprotein-cholesterol ratios for various distribution and chemical composition of ultraanimal species have been obtained employing cellucentrifugally separated lipoproteins in human serum. J. lose acetate electrophoretic separaton of lipoproteins clin. Incest. 34, 1345-1353. coupled with an enzymatic cholesterol stain. The Johansson M. B. and Karlsson B. W. (1976) Lipoproteins in serum of rat, mouse, gerbil, rabbit, pig and man studied L D L - C / H D L - C ratios were presently found to be by electrophoretical and immunological methods. Comp. markedly higher in the rabbit, monkey and human, Biochem. Physiol. 54B, 495-500. species known to be more prone to experimentallyinduced atherosclerotic lesions (Beher et al., 1963; Koga S., Horwitz D. L. and Scanu A. M. (1969) Isolation and properties of lipoproteins from normal rat serum. J. Mahley et al., 1974) than the L D L - C / H D L - C ratios Lipid Res. 10, 577-588. of the rat, mouse and dog, which are species less Kramsch D. M., Aspen A. J. and Rozler L. J. (1981) prone to induced atherosclerotic lesions (Wissler et Atherosclerosis: prevention by agents not affectingabnoraL, 1954; Fillios et al., 1956; Camejo et al., 1973; mal levels of blood lipids. Science 213, 1511-1512. Mahley, 1978; Kramsch et al., 1981). The total Mahley R. W. (1978) Disturbances in Lipid and Lipoprotein cholesterol to H D L - C ratio was also found to be Metabolism, pp. 181-197. Williams and Wilkins, Baltimore, MD. higher in those species most prone to induced atherosclerotic lesions, although the species differences be- Mahley R. W., Weisgraber K. H. and Innerarity T. (1974) Canine lipoproteins and atherosclerosis II. Charactertween the total cholesterol/HDL-C ratios appear to ization of the plasma lipoproteins associated with atherobe less marked than the species differences between genic and nonatherogenic hyperlipidemia. Circulation the L D L ~ 2 / H D L - C ratios. Human epidemiological Res. 35, 722-733. evidence has shown that the L D L - C / H D L - C ratio is Manual of Laboratory Operations (1974) Lipid Research positively correlated to the risk of developing atheroClinics Programs. DHEW Publication No. (NIH) 75-628. sclerotic disease (Chapman, 1980), therefore it is Narayan K. A. (197I) Lowered serum concentration of high possible that a high L D L - C / H D L - C ratio may be an density lipoproteins in cholesterol-fed rats. Atherosclerosis 13, 205-215. important indicator of species susceptability to the experimental induction of atherosclerotic lesions. In Oschry Y. and Eisenberg S. (1982) Rat plasma lipoproteins: reevaluation of a lipoprotein system in an animal devoid addition, since it has been reported that cholesterol of cholesterol ester transfer activity. J. Lipid Res. 23, ester transfer does not occur between rat plasma 1099-1106. lipoproteins (Oschry and Eisenberg, 1982) the possi- Schjeide O. A., Rivin A. U. and Yoshino J. (1963) Uptake bility exists that rat prealbumin lipoprotein (PAL) of lipid stains by lipids and serum lipoproteins. Am. J. may have a role in the clearance of cholesterol from clin, Path. 39, 329 341. rat plasma and experiments are currently in progress Steele B. W., Koehler D. F., Azar M. M., Blaszkowski T.
44
JOSEPH R. DAVIS et al. P., Kuba K. and Dempsey M. E. (1976) Enzymatic determination of cholesterol in high-density-lipoprotein fractions prepared by a precipitation technique. Clin. Chem. 22, 98-101.
Wissler R. W., Eilert M. L., Schroeder M. A. and Cohen L. (1954) Production of lipomatous and atheromatous arterial lesions in the albino rat. A. M. A. Archs Path. 57, 333-351.