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Isolation and characterization of the major lipoprotein density classes of normal and diabetic baboon (Papio anubis) plasma
481
Atherosclerosis, 31 (1978) 481-487 @ Elsevier/North-Holland Scientific Publishers, Ltd.
Preliminary ______.
Note
I_-~--
ISOLATION AND CHARACTERIZATION OF THE MAJOR LIPOPROTEIN DENSITY CLASSES OF NORMAL AND DIABETIC BABOON (PAPIO ANUBZS) PLASMA
DUB0 BOJANOVSKI
*, PETAR ALAUPOVIC,
JIM L. KELLEY and CLARKE STOUT
Institut fur Klinische Biochemie und Physiologische Chemie der Medizinischen Hochschule, Hannover (G.F.R.); Laboratory of Lipid and Lipoprotein Studies, Oklahoma Medical Research Foundation, and Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104; and Department of Pathology, The University of Texas Medical Branch, Galveston, TX 77550 (U.S.A.) (Received 15 March, 1978) (Revised, received 18 August, 1978) (Accepted 23 August, 1978)
Summary Lipoproteins from the plasma of normal and diabetic baboons (Papio an&is) were separated into very low density lipoproteins (VLDL, d 1.006 g/ml), low density lipoproteins (LDL, d 1.006-1.063 g/ml) and high density lipoproteins (HDL, d 1.063-1.21 g/ml) and the density classes were characterized by immunologic methods and by quantitative determination of protein and lipid. Diabetes was induced by surgical removal of the pancreas and the hyperglycemia was controlled by daily insulin injection. HDL were the major density class of normal baboon plasma representing 66% of the total lipoprotein content. LDL accounted for 31% and the VLDL for only 3%. Diabetes had a profound effect on the distribution of baboon lipoproteins. VLDL accounted for 37%, LDL for 35% and HDL for only 28% of the total lipoprotein content. In normal baboons, HDL was the main vehicle for the transport of phospholipids and glycerides with cholesterol almost equally distributed between HDL and LDL. In diabetic animals, free and esterified cholesterol were transported mainly by LDL and the glycerides by VLDL. Cholesterol esters accounted for approximately 75% of the total cholesterol in both the normal and diabetic baboons. Studies on the immunologic properties of baboon lipoproteins showed a This work was supported in part by grants HL-6221 and HL-1005 from the U.S. Public Health Service, a grant No. 76227 from the John A. Hartford Foundation. and the resources of the Oklahoma Medical Research Foundation. * D. Bojanovski was recipient of a NATO-Fellowship (430/402/859/4) from the Deutscher Akademischer Austauschdienst.
482
marked heterogeneity of all 3 major density classes with respect to antigenic composition. Antisera to human apolipoproteins A (A-I and A-II), B, C (C-I, C-II and C-III), D and E cross-reacted with baboon lipoproteins suggesting a close analogy between apolipoproteins of these two species. Key words:
Apolipoproteins - High density lipoproteins - Immunodiffusion sity lipoproteins - Very low density lipoproteins
- Low den-
Introduction The baboon (Papio an&is) has been selected as the representative of nonhuman primates for our studies on the comparative biochemistry of plasma lipoprotein systems. They develop aortic atherosclerosis in the natural environment [l-3] and respond to cholesterol supplemented diets with increased serum lipid levels and aortic lesions [3-71. Baboons have already been utilized as experimental animals for investigating human arterial disease [4,5,8] and, because of their physiologic and phylogenetic similarity to man, baboons may also be used as a convenient model for studying the normal and deranged lipid transport processes [9 1. Lack of data on the chemical and immunochemical properties of plasma apolipoproteins and lipoproteins seems to be one of the major restrictions for a wider use of baboons as a model for studying atherosclerosis and abnormalities of lipid metabolism. The present paper describes the chemical and immunologic composition of the major lipoprotein density classes from normal and diabetic baboons. Materials and Methods Blood from normal and diabetic baboons was obtained under ketamine hydrochloride sedation. Approximately 100 ml of blood was aspirated from the femoral vein of each animal into heparinized plastic syringes, transferred into heparinized glass tubes, centrifuged and the plasma was transferred into clean plastic containers. Plasma was shipped immediately to Oklahoma City from Galveston in Styrofoam boxes kept cool by the addition of “Koolits”. Diabetes was induced by surgical removal of the pancreas from 14 female young adult and adult baboons (Papio anubis). This procedure resulted in immediate and permanent hyperglycemia and glycosuria. The hyperglycemia was controlled by daily intramuscular injections of insulin, the dosage of which was adjusted shortly after pancreatectomy to obtain morning urine glucose concentrations of 4+ (poor diabetic control). Urine glucose concentration was measured using Clinitest tablets (donated by the Ames Company). Blood samples were taken from baboons which had been diabetic for an average duration of 52 months (range 44-59 months). The levels of plasma glucose and lipids from normal and diabetic animals are shown in Table 1. The baboons were fed a semi-synthetic diet containing 17% protein, 5% fat, 3% fiber and fresh fruit. Lipoprotein density classes were separated by preparative ultracentrifugation
483
TABLE
1
LIPID
AND
GLUCOSE
CONCENTRATIONS
n
Animals
Total
Glucose (mg/lOO
Normal
14
Diabetic
14
64f 457
IN NORMAL
ml) 34a
+ 106
AND cholesterol
(mg/lOO 104
f 18
144
? 50
ml)
DIABETIC
BABOON
PLASMA
Triglyceride (mg/lOO 60t 255
ml) 32
+ 172
___ a Mean
f standard
deviation.
as described previously [lo] into very low density lipoproteins (VLDL, d < 1.006 g/ml), low density lipoproteins (LDL, d 1.006-1.063 g/ml), and high density lipoproteins (HDL, d 1.063-1.21 g/ml). In some experiments HDL were subfractionated into HDLl (d 1.063-1.125 g/ml) and HDL3 (d 1.1251.21 g/ml). The density classes were recentrifuged until no albumin was detected by double diffusion analysis. Delipidation and chemical analyses have been described in detail previously [lo]. Double immunodiffusion [ll] analyses were performed in 1% agarose gels employing Veronal buffer, pH 8.5, ionic strength 0.05. Monospecific antisera to human apolipoproteins A-I, A-II, C-I, C-II, C-III, D and E, and to lipoproteins A and B (LP-A and LP-B) were prepared and tested as described in previous reports [ 12-151. Results Distribution and chemical composition of lipoprotein density classes As shown in Table 2, the HDL were the main density class of normal baboon plasma; they accounted for 66% of the total lipoprotein content. The LDL accounted for 31% and the VLDL for only 3%. The distribution of density classes in diabetic animals was characterized by an absolute increase in VLDL and LDL and a decrease of HDL, compared to normal baboons. In normal baboons, the HDL density class was the main vehicle for the transport of phospholipids and glycerides on a weight basis. The unesterified cholesterol and cholesterol esters were almost equally distributed between HDL and LDL (Table 2). In diabetic animals, the unesterified and esterified cholesterol were found mainly in LDL and the triglycerides in VLDL. The triglyceride content of diabetic VLDL was much higher than that found in normal VLDL. Cholesterol esters represented approximately 75% of the total plasma cholesterol in both the normal and diabetic baboons. Although in diabetic animals there is a reduction of total apolipoprotein content, the apoprotein levels of VLDL and LDL are higher than in normal animals. Immunologic properties of lipoprotein density classes Results of immunologic characterization of baboon density classes with monospecific antisera to human apolipoproteins or their constitutive polypeptides are shown in Table 3. Both the normal and diabetic VLDL reacted positively with antibodies to LP-B and ApoD. In addition, diabetic VLDL reacted with antibodies to apolipoproteins A-I, A-II, C-I, C-II, C-III and D. Due to the
24.0 f 3.10 41.7 + 4.30 64.6 + 25.2
Diabetic plasma VLDL LDL HDL
13.2 t 2.81 28.3 ? 4.33 19.6 + 3.91
0.7 f 0.15 16.2 + 3.34 55.0 f 5.31
Phospholipid
5.8 + 0.23 23.4 f 6.75 11.9 + 1.12
1.1 t 0.10 15.6 f 3.30 13.2 f 2.10
Free
0.17 5.80 1.65
PLASMA
16.5 +_ 4.32 59.0 + 12.45 19.6 +_ 2.27
3.0 ? 47.8 f 36.5 ?
Ester
LIPIDS OF BABOON
Cholesterol
AND INDIVIDUAL
0.17 0.80 0.65
124.8 +_21.34 23.1 t 1.10 20.9 ? 1.63
5.0 f 16.8 f 29.3 +
Glycerides
LIPOPROTEIN
CLASSES
184.30 175.50 136.55
10.83 128.10 267.70
Lipoprotein
DENSITY
(37.13%) (35.36%) (27.57%)
(2.66%) (31.50%) (65.83%)
b
a Values represent the mean + standard deviation of 4 independent pools of plasma. Plasma was pooled from several animals for each isolation of lipoprotein density classes. b Values in parentheses represent the percent of the total lipoprotein content in each lipoprotein density &us.
Normal
1.02 O.lsa 31.7 + 11.60 133.7 f 21.20
(mg/lOO ml)
Protein
OF APOLIPOPROTEINS
plasma VLDL LDL HDL
Density class
CONCENTRATIONS
TABLE 2
485 TABLE
3
THE ANTIGENIC COMPOSITION DIABETIC BABOON PLASMA a Density class
nVLDLb d VLDL n LDL d LDL n HDL d HDL d HDL2 d HDL3
OF
LIPOPROTEIN
DENSITY
CLASSES
OF
NORMAL
Antisera
to human
apolipoproteins
A-I
A-II
B
C-I
C-II
C-III
D
E
Albumin
+ + + + + + +
+ + + + + + +
+ + + + + + + -
+
-
+ _ -
+ + + + + + + +
+ + + + +
-
N.D.’ N.D.
-
+ 5 * ?r
k ? f + *
+ + + +
AND
-
a Sign + represents a distinct immunoprecipitin line or arc obtained in 1% agarose gel by immunodiffusion and/or immunoelectrophoresis. Sign f indicates that a precipitin line was observed with some. but not all lipoprotein preparations. Sign - indicates that a precipitin line was never observed. b n = normal animals, d = diabetic animals. ’ N.D. = not determined.
very low protein content of normal VLDL, the ApoC was detected in VLDL from diabetic but not in VLDL from normal baboons. The LDL from normal and diabetic baboons gave positive reactions with antibodies to apolipoproteins B, A-I, A-II, D and E. Both the normal and diabetic HDL showed positive immunoprecipitin lines with all human antisera tested. The only difference between HDL subfractions was the absence of ApoB in HDL3. Discussion High density lipoproteins are the major circulating lipoprotein density class in plasma of normal baboons. In this respect, baboons resemble a number of avian [10,16] and mammalian species 1171 which, unlike man, are characterized by high levels of HDL and low levels of LDL. The lipoprotein system of normal baboons is further characterized by the virtual absence of chylomicrons and a very low concentration of VLDL. The concentrations of VLDL and LDL are comparable to those reported for pre-@- and P-lipoproteins in normal baboons [ 181. Studies on the chemical and immunochemical characterization of the plasma lipoprotein system of baboons showed clearly a marked heterogeneity of all 3 major density classes with respect to chemical and antigenic composition. The LDL were characterized by ApoB and the HDL by ApoA as the major protein components. However, the A-I and A-II polypeptides were also detected in LDL. The C-I, C-II and C-III polypeptides and ApoD and ApoE were consistently detected immunochemically in LDL and HDL. These results suggest that, in analogy to the human lipoprotein system [12,13,19], baboon lipoproteins also consist of a mixture of polydisperse lipoprotein families. Due to the paucity of data in the literature, it is still not possible to compare systematically the plasma lipoprotein system of non-human primates. It seems,
486
however, that there are some significant differences between species both with respect to the distribution and composition of major density classes. Similarly to Erythrocebus patas monkey [17,20] and Rhesus monkey [21], the baboon lipoprotein system is also characterized by a high concentration of HDL accounting for almost 70% of the total lipoprotein content. In contrast, the HDL of chimpanzee only account for 47% of the total lipoprotein content 1221. A low ratio of CX-and P-lipoprotein cholesterol was also observed in the spider monkey and the macaque [ 181. The major difference in the chemical composition between density classes of baboon and Patas monkey [20] pertains to the phospholipid and cholesterol content. The VLDL, LDL and HDL of Patas monkey have higher content of phospholipid and lower content of cholesterol than the corresponding density classes of baboon. On the other hand, there were no remarkable differences in the chemical composition between LDL of baboon and Rhesus [23] monkeys or between HDL of baboon and either Rhesus [ 211 or chimpanzee [ 221. Experimental induction of diabetes has a profound effect on the baboon lipoprotein system. In diabetic baboons the concentrations of VLDL and LDL increase and the concentration of HDL decreases resulting in a dramatic change in the distribution of density classes compared to the normal baboon. Whereas in normal baboon the HDL accounted for almost 70% of the total lipoproteins, in diabetic baboons the HDL only accounted for 30%. Another major change was an increase in the VLDL which in normal animals represented barely 2% but in diabetic animals accounted for 38% of the total lipoprotein content. These results are similar to those observed in human subjects with poorly controlled diabetes mellitus, i.e., elevation of VLDL and reduction of HDL [ 241. The most significant finding of this study is the cross-reactivity of antisera to human apolipoproteins with baboon apolipoproteins suggesting a close analogy between apolipoproteins A, B, C, D and E of these two species. Furthermore, the distribution of apolipopoteins throughout the density spectrum is very similar in both species. Results of this study on the analogy between baboon and human apolipoproteins and lipoproteins and on changes in the lipoprotein system caused by experimentally-induced diabetes represent further evidence for the usefulness of the baboon as an experimental model for studying the lipid transport processes in normal and deranged metabolic states. Acknowledgements We wish to thank Mrs. C. Beaver for her help in the preparation script. Insulin was donated by E.R. Squibb and Sons.
of the manu-
References 1 McGill, Jr., H.C., Strong, J.C., Holman. R.L. and Werthenssen. N.T., Arterial lesions in the Kenya baboon, Circulation Res., 8 (1960) 670. 2 Lindsay, S. and Chaikoff, I.L., Naturally occurring arteriosclerosis in nonhuman primates, J. Atheros&r. Res.. 6 (1966) 36. 3 Howard. A.N., Gresham, G.A., Bowyer, D.E. and Lindgren. F.T.. Aortic and coronary atherosclerosis in baboons, Progr. Biochem. Pharmacol., 4 (1968) 438.
4 Strong,
J.P.
and McGill, Jr.,
H.C.,
Diet and experimental
atherosclerosis
in baboons,
Amer.
.J. Path..
50 (1967) 669. 5 Blaton, V., Howard, A.N.. Gresham, G.A.. Vandamine. D. and Peeters, H., Lipid changes in the plasma lipoproteins of baboons given an atherogenic diet. Part 1 (Changes in the lipids of total plasma and aand P-lipoproteins), Atherosclerosis, 11 (1970) 497. 6 Eggen, D.A. and Strong, J.P., Diet and cholesterol metabolism in young and old baboons. Atherosclerosis, 12 (1970) 359. 7 Kotze. J.P. and Kempff, P.G.. Nutritional regimens and their effects on the lipid metabolism of the baboon (Papio ursinus), S. Afr. J. Sci., 69 (1973) 371. 8 McGill. Jr., H.C., Strong, J.P., Newman, W.P. and Eggen. D.A., The baboon in atherosclerosis research. In: H. Vogtborg (Ed.), The Baboon in Medical Research, Vol. II, University of Texas Press, Austm, TX, 1967. p. 351. 9 Shapiro, I.L., Jastremsky. J.A.. Kritchevsky. D. and Eggen. D.A., Transport of cholesterol in the baboon. In: H. Vogtborg (Ed.), The Baboon in Medical Research, Vol. II, University of Texas Press. Austin, TX, 1967, p. 571. 10 Kelley, J.L. and Alaupovic, P., Lipid transport in the avian species, Part 1 (Isolation and characterization of apolipoproteins and major lipoprotein density classes of male turkey serum), Atherosclerosis, 24 (1976) 155. 11 Ouchterlony, 6.. Antigen-antibody reactions in gels, Part 4 (Types of reactions in coordinated systems of diffusion), Acta Path. Microbial.. 32 (1953) 231. 13 Alaupovic. P.. Lee. D.M. and McConathy, W.J., Studies on the composition and structure of plasma lipoproteins -- Distribution of lipoprotein families in major density classes of normal human plasma lipoproteins, Biochim. Biophys. Acta 260 (1972) 689. 13 Kostner. G. and Alaupovic, P., Studies of the composition and structure of plasma lipoproteins -Separation and quantification of the lipoprotein families occurring in the high density lipoproteins of human plasma. Biochemistry, 11 (1972) 3419. 14 McConathy, W.J. and Alaupovic. P., Studies on the isolation and partial characterization of apolipoprotein D and lipoprotein D of human plasma, Biochemistry. 15 (1976) 515. 15 Curry, M.D., McConathy, W.J.. Alaupovic. P.. Ledford, J.H. and Popovid. M., Determination of human apolipoprotein E by electroimmunoassay, Biochim. Biophys. Acta, 439 (1976) 413. 16 Mills, G.L. and Taylour. C.E., The distribution and composition of serum lipoproteins in eighteen animals, Comp. Biochem. Physiol., 40-B (1971) 489. 17 Peeters, H.. Blaton, V., Declercq, B.. Howard, A.N. and Gresham, G.A.. Lipid changes in the plasma lipoproteins of baboons given an atherogenic diet, Part 2 (Changes in the phospholipid classes of total plasma and of cy- and /3-lipoproteins), Atherosclerosis, 12 (1970) 283. 18 Srinivasan, S.R., McBride, Jr., J.R.. Radhakrishnamurthy, B. and Berenson. G.S., Comparative studies on serum lipoprotein and lipid profiles in subhuman primates, Comp. Biochem. Physiol.. 47-B (1974) 711. 19 Alaupovic. P., Conceptual development of the classification systems of plasma lipoproteins. In: H. Peeters (Ed.), Protides of the Biological Fluids (Proc. Colloq.. Vol. 19), Pergamon Press, London, 1972. P. 9. 20 Mahley, R.W., Weisgraber, K.H., Innerarity, T. and Brewer, Jr., H.B., Characterization of the plasma lipoproteins and apoproteins of the Erythrocebus patas monkey. Biochemistry, 15 (1976) 1928. 21 Scanu. A.M.. Edelstein. C., Vitello, L., Jones, R. and Wissler, R., The serum high density lipoproteins of Macaws rhesus, Part 1 (Isolation, composition and properties), J. Biol. Chem., 248 (1973) 7648. 22 Blaton. V.. Vercaemst, R., Vandecasteele. N.. Caster, H. and Peeters, H., Isolation and partial characterization of chimpanzee plasma high density lipoproteins and their apolipoproteins, Biochemistry, 13 (1974) 1127. 23 Lee, J.A. and Morris, M.D., Characterization of the serum low density lipoproteins of normal on two Rhesus monkeys with spontaneous hyperbeta-lipoproteinemia. Biochem. Med., 10 (1974) 245. 24 Hamwi. G.J., Garcia. O., Kruger, F.A., Gwinup. G. and Cornwell, D.G.. Hyperlipidemia in uncontrolled diabetes,
Metabolism,
11 (1972)
850.
Atherosclerosis, 31 (1978) 481-487 @ Elsevier/North-Holland Scientific Publishers, Ltd.
Preliminary ______.
Note
I_-~--
ISOLATION AND CHARACTERIZATION OF THE MAJOR LIPOPROTEIN DENSITY CLASSES OF NORMAL AND DIABETIC BABOON (PAPIO ANUBZS) PLASMA
DUB0 BOJANOVSKI
*, PETAR ALAUPOVIC,
JIM L. KELLEY and CLARKE STOUT
Institut fur Klinische Biochemie und Physiologische Chemie der Medizinischen Hochschule, Hannover (G.F.R.); Laboratory of Lipid and Lipoprotein Studies, Oklahoma Medical Research Foundation, and Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104; and Department of Pathology, The University of Texas Medical Branch, Galveston, TX 77550 (U.S.A.) (Received 15 March, 1978) (Revised, received 18 August, 1978) (Accepted 23 August, 1978)
Summary Lipoproteins from the plasma of normal and diabetic baboons (Papio an&is) were separated into very low density lipoproteins (VLDL, d 1.006 g/ml), low density lipoproteins (LDL, d 1.006-1.063 g/ml) and high density lipoproteins (HDL, d 1.063-1.21 g/ml) and the density classes were characterized by immunologic methods and by quantitative determination of protein and lipid. Diabetes was induced by surgical removal of the pancreas and the hyperglycemia was controlled by daily insulin injection. HDL were the major density class of normal baboon plasma representing 66% of the total lipoprotein content. LDL accounted for 31% and the VLDL for only 3%. Diabetes had a profound effect on the distribution of baboon lipoproteins. VLDL accounted for 37%, LDL for 35% and HDL for only 28% of the total lipoprotein content. In normal baboons, HDL was the main vehicle for the transport of phospholipids and glycerides with cholesterol almost equally distributed between HDL and LDL. In diabetic animals, free and esterified cholesterol were transported mainly by LDL and the glycerides by VLDL. Cholesterol esters accounted for approximately 75% of the total cholesterol in both the normal and diabetic baboons. Studies on the immunologic properties of baboon lipoproteins showed a This work was supported in part by grants HL-6221 and HL-1005 from the U.S. Public Health Service, a grant No. 76227 from the John A. Hartford Foundation. and the resources of the Oklahoma Medical Research Foundation. * D. Bojanovski was recipient of a NATO-Fellowship (430/402/859/4) from the Deutscher Akademischer Austauschdienst.
482
marked heterogeneity of all 3 major density classes with respect to antigenic composition. Antisera to human apolipoproteins A (A-I and A-II), B, C (C-I, C-II and C-III), D and E cross-reacted with baboon lipoproteins suggesting a close analogy between apolipoproteins of these two species. Key words:
Apolipoproteins - High density lipoproteins - Immunodiffusion sity lipoproteins - Very low density lipoproteins
- Low den-
Introduction The baboon (Papio an&is) has been selected as the representative of nonhuman primates for our studies on the comparative biochemistry of plasma lipoprotein systems. They develop aortic atherosclerosis in the natural environment [l-3] and respond to cholesterol supplemented diets with increased serum lipid levels and aortic lesions [3-71. Baboons have already been utilized as experimental animals for investigating human arterial disease [4,5,8] and, because of their physiologic and phylogenetic similarity to man, baboons may also be used as a convenient model for studying the normal and deranged lipid transport processes [9 1. Lack of data on the chemical and immunochemical properties of plasma apolipoproteins and lipoproteins seems to be one of the major restrictions for a wider use of baboons as a model for studying atherosclerosis and abnormalities of lipid metabolism. The present paper describes the chemical and immunologic composition of the major lipoprotein density classes from normal and diabetic baboons. Materials and Methods Blood from normal and diabetic baboons was obtained under ketamine hydrochloride sedation. Approximately 100 ml of blood was aspirated from the femoral vein of each animal into heparinized plastic syringes, transferred into heparinized glass tubes, centrifuged and the plasma was transferred into clean plastic containers. Plasma was shipped immediately to Oklahoma City from Galveston in Styrofoam boxes kept cool by the addition of “Koolits”. Diabetes was induced by surgical removal of the pancreas from 14 female young adult and adult baboons (Papio anubis). This procedure resulted in immediate and permanent hyperglycemia and glycosuria. The hyperglycemia was controlled by daily intramuscular injections of insulin, the dosage of which was adjusted shortly after pancreatectomy to obtain morning urine glucose concentrations of 4+ (poor diabetic control). Urine glucose concentration was measured using Clinitest tablets (donated by the Ames Company). Blood samples were taken from baboons which had been diabetic for an average duration of 52 months (range 44-59 months). The levels of plasma glucose and lipids from normal and diabetic animals are shown in Table 1. The baboons were fed a semi-synthetic diet containing 17% protein, 5% fat, 3% fiber and fresh fruit. Lipoprotein density classes were separated by preparative ultracentrifugation
483
TABLE
1
LIPID
AND
GLUCOSE
CONCENTRATIONS
n
Animals
Total
Glucose (mg/lOO
Normal
14
Diabetic
14
64f 457
IN NORMAL
ml) 34a
+ 106
AND cholesterol
(mg/lOO 104
f 18
144
? 50
ml)
DIABETIC
BABOON
PLASMA
Triglyceride (mg/lOO 60t 255
ml) 32
+ 172
___ a Mean
f standard
deviation.
as described previously [lo] into very low density lipoproteins (VLDL, d < 1.006 g/ml), low density lipoproteins (LDL, d 1.006-1.063 g/ml), and high density lipoproteins (HDL, d 1.063-1.21 g/ml). In some experiments HDL were subfractionated into HDLl (d 1.063-1.125 g/ml) and HDL3 (d 1.1251.21 g/ml). The density classes were recentrifuged until no albumin was detected by double diffusion analysis. Delipidation and chemical analyses have been described in detail previously [lo]. Double immunodiffusion [ll] analyses were performed in 1% agarose gels employing Veronal buffer, pH 8.5, ionic strength 0.05. Monospecific antisera to human apolipoproteins A-I, A-II, C-I, C-II, C-III, D and E, and to lipoproteins A and B (LP-A and LP-B) were prepared and tested as described in previous reports [ 12-151. Results Distribution and chemical composition of lipoprotein density classes As shown in Table 2, the HDL were the main density class of normal baboon plasma; they accounted for 66% of the total lipoprotein content. The LDL accounted for 31% and the VLDL for only 3%. The distribution of density classes in diabetic animals was characterized by an absolute increase in VLDL and LDL and a decrease of HDL, compared to normal baboons. In normal baboons, the HDL density class was the main vehicle for the transport of phospholipids and glycerides on a weight basis. The unesterified cholesterol and cholesterol esters were almost equally distributed between HDL and LDL (Table 2). In diabetic animals, the unesterified and esterified cholesterol were found mainly in LDL and the triglycerides in VLDL. The triglyceride content of diabetic VLDL was much higher than that found in normal VLDL. Cholesterol esters represented approximately 75% of the total plasma cholesterol in both the normal and diabetic baboons. Although in diabetic animals there is a reduction of total apolipoprotein content, the apoprotein levels of VLDL and LDL are higher than in normal animals. Immunologic properties of lipoprotein density classes Results of immunologic characterization of baboon density classes with monospecific antisera to human apolipoproteins or their constitutive polypeptides are shown in Table 3. Both the normal and diabetic VLDL reacted positively with antibodies to LP-B and ApoD. In addition, diabetic VLDL reacted with antibodies to apolipoproteins A-I, A-II, C-I, C-II, C-III and D. Due to the
24.0 f 3.10 41.7 + 4.30 64.6 + 25.2
Diabetic plasma VLDL LDL HDL
13.2 t 2.81 28.3 ? 4.33 19.6 + 3.91
0.7 f 0.15 16.2 + 3.34 55.0 f 5.31
Phospholipid
5.8 + 0.23 23.4 f 6.75 11.9 + 1.12
1.1 t 0.10 15.6 f 3.30 13.2 f 2.10
Free
0.17 5.80 1.65
PLASMA
16.5 +_ 4.32 59.0 + 12.45 19.6 +_ 2.27
3.0 ? 47.8 f 36.5 ?
Ester
LIPIDS OF BABOON
Cholesterol
AND INDIVIDUAL
0.17 0.80 0.65
124.8 +_21.34 23.1 t 1.10 20.9 ? 1.63
5.0 f 16.8 f 29.3 +
Glycerides
LIPOPROTEIN
CLASSES
184.30 175.50 136.55
10.83 128.10 267.70
Lipoprotein
DENSITY
(37.13%) (35.36%) (27.57%)
(2.66%) (31.50%) (65.83%)
b
a Values represent the mean + standard deviation of 4 independent pools of plasma. Plasma was pooled from several animals for each isolation of lipoprotein density classes. b Values in parentheses represent the percent of the total lipoprotein content in each lipoprotein density &us.
Normal
1.02 O.lsa 31.7 + 11.60 133.7 f 21.20
(mg/lOO ml)
Protein
OF APOLIPOPROTEINS
plasma VLDL LDL HDL
Density class
CONCENTRATIONS
TABLE 2
485 TABLE
3
THE ANTIGENIC COMPOSITION DIABETIC BABOON PLASMA a Density class
nVLDLb d VLDL n LDL d LDL n HDL d HDL d HDL2 d HDL3
OF
LIPOPROTEIN
DENSITY
CLASSES
OF
NORMAL
Antisera
to human
apolipoproteins
A-I
A-II
B
C-I
C-II
C-III
D
E
Albumin
+ + + + + + +
+ + + + + + +
+ + + + + + + -
+
-
+ _ -
+ + + + + + + +
+ + + + +
-
N.D.’ N.D.
-
+ 5 * ?r
k ? f + *
+ + + +
AND
-
a Sign + represents a distinct immunoprecipitin line or arc obtained in 1% agarose gel by immunodiffusion and/or immunoelectrophoresis. Sign f indicates that a precipitin line was observed with some. but not all lipoprotein preparations. Sign - indicates that a precipitin line was never observed. b n = normal animals, d = diabetic animals. ’ N.D. = not determined.
very low protein content of normal VLDL, the ApoC was detected in VLDL from diabetic but not in VLDL from normal baboons. The LDL from normal and diabetic baboons gave positive reactions with antibodies to apolipoproteins B, A-I, A-II, D and E. Both the normal and diabetic HDL showed positive immunoprecipitin lines with all human antisera tested. The only difference between HDL subfractions was the absence of ApoB in HDL3. Discussion High density lipoproteins are the major circulating lipoprotein density class in plasma of normal baboons. In this respect, baboons resemble a number of avian [10,16] and mammalian species 1171 which, unlike man, are characterized by high levels of HDL and low levels of LDL. The lipoprotein system of normal baboons is further characterized by the virtual absence of chylomicrons and a very low concentration of VLDL. The concentrations of VLDL and LDL are comparable to those reported for pre-@- and P-lipoproteins in normal baboons [ 181. Studies on the chemical and immunochemical characterization of the plasma lipoprotein system of baboons showed clearly a marked heterogeneity of all 3 major density classes with respect to chemical and antigenic composition. The LDL were characterized by ApoB and the HDL by ApoA as the major protein components. However, the A-I and A-II polypeptides were also detected in LDL. The C-I, C-II and C-III polypeptides and ApoD and ApoE were consistently detected immunochemically in LDL and HDL. These results suggest that, in analogy to the human lipoprotein system [12,13,19], baboon lipoproteins also consist of a mixture of polydisperse lipoprotein families. Due to the paucity of data in the literature, it is still not possible to compare systematically the plasma lipoprotein system of non-human primates. It seems,
486
however, that there are some significant differences between species both with respect to the distribution and composition of major density classes. Similarly to Erythrocebus patas monkey [17,20] and Rhesus monkey [21], the baboon lipoprotein system is also characterized by a high concentration of HDL accounting for almost 70% of the total lipoprotein content. In contrast, the HDL of chimpanzee only account for 47% of the total lipoprotein content 1221. A low ratio of CX-and P-lipoprotein cholesterol was also observed in the spider monkey and the macaque [ 181. The major difference in the chemical composition between density classes of baboon and Patas monkey [20] pertains to the phospholipid and cholesterol content. The VLDL, LDL and HDL of Patas monkey have higher content of phospholipid and lower content of cholesterol than the corresponding density classes of baboon. On the other hand, there were no remarkable differences in the chemical composition between LDL of baboon and Rhesus [23] monkeys or between HDL of baboon and either Rhesus [ 211 or chimpanzee [ 221. Experimental induction of diabetes has a profound effect on the baboon lipoprotein system. In diabetic baboons the concentrations of VLDL and LDL increase and the concentration of HDL decreases resulting in a dramatic change in the distribution of density classes compared to the normal baboon. Whereas in normal baboon the HDL accounted for almost 70% of the total lipoproteins, in diabetic baboons the HDL only accounted for 30%. Another major change was an increase in the VLDL which in normal animals represented barely 2% but in diabetic animals accounted for 38% of the total lipoprotein content. These results are similar to those observed in human subjects with poorly controlled diabetes mellitus, i.e., elevation of VLDL and reduction of HDL [ 241. The most significant finding of this study is the cross-reactivity of antisera to human apolipoproteins with baboon apolipoproteins suggesting a close analogy between apolipoproteins A, B, C, D and E of these two species. Furthermore, the distribution of apolipopoteins throughout the density spectrum is very similar in both species. Results of this study on the analogy between baboon and human apolipoproteins and lipoproteins and on changes in the lipoprotein system caused by experimentally-induced diabetes represent further evidence for the usefulness of the baboon as an experimental model for studying the lipid transport processes in normal and deranged metabolic states. Acknowledgements We wish to thank Mrs. C. Beaver for her help in the preparation script. Insulin was donated by E.R. Squibb and Sons.
of the manu-
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