Intestinal lymph and plasma lipoproteins in the preruminant calf: partial resolution of particle heterogeneity in the 1.040-1.090 g/ml interval.

Our previous studies in the preruminant calf have provided evidence for the heterogeneity of lipoprotein particles in the 1.040-1.090 g/ml density interval in both plasma and postprandial intestinal lymph (Bauchart, D. et al., 1989. J. Lipid Res. 30: 1499-1514; and Laplaud, P. M. et al., 1990. J. Lipid Res. 31: 1781-1792). We therefore attempted to resolve this heterogeneity by use of heparin-Sepharose affinity chromatography. Experiments were performed on three calves; portal vein plasma and intestinal lymph were obtained simultaneously 10 h after a meal, i.e., at peak lipid absorption. In both fluids, the chromatographic profile presented three fractions, I, II, and III. Fraction I was characterized by the presence of cholesteryl ester-rich particles (approximately 35-37% of lipoprotein mass), which migrated electrophoretically as typical high density lipoproteins and exhibited Stokes diameters in the 130-160 A range; apoA-I was the predominant protein. In addition to this polypeptide, fraction II contained small amounts of a supplementary protein (Mr approximately 51,000), exhibiting heparin-binding properties. In the light of results reported in the literature, we suggest that this latter protein could correspond to beta 2 glycoprotein I. The chemical composition of each fraction II closely resembled that of the corresponding fraction I, while their electrophoretic migrations appeared slightly slower and their Stokes diameters slightly larger (155-165 A). Apart from the presence of small amounts of apoA-I, two high Mr proteins (Mr approx. 560,000 and 300,000) were typical of the apolipoprotein moiety of fractions III. The lower Mr form was present as a trace component only in fraction III originating from plasma; its proportion increased in lymph fraction III so as to approximately match that of the higher Mr (i.e., 560,000) protein. In both plasma and lymph, fraction III was electrophoretically heterogeneous, exhibiting a doublet of bands with migration and Stokes diameters (250 A) typical of low density lipoprotein particles. However, no evidence for the presence of a particle resembling lipoprotein[a] in fraction III could be obtained. In lymph only, fraction III contained a supplementary population of lipoproteins with migration intermediary between those of conventional low and high density lipoproteins and with Stokes diameters in the 190-200 A range. Other specific features of lymph fraction III included a sevenfold increase in its triglyceride content (8.5 +/- 3.4% vs. 1.2 +/- 1.1% in the corresponding fraction from plasma), to the detriment of cholesteryl esters, and a higher proportion of protein.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization and amino-terminal sequence of apolipoprotein AI from plasma high density lipoproteins in the preruminant calf, Bos spp.

The major apolipoprotein of calf plasma high-density lipoproteins, apo-AI, has been isolated and characterized. Apolipoprotein AI (apo-AI) was separated from the protein moiety of high-density lipoproteins (d 1.090-1.180 g/ml) by preparative electrophoresis in SDS-polyacrylamide gels followed by electrophoretic elution. Purified calf apo-AI had an Mr of approx. 27,000-28,000 in SDS-polyacrylamide gels, resembling human apo-AI. The amino acid composition of calf apo-AI displayed an overall similarity to that of its human and other mammalian counterparts (baboon, dog, badger, rabbit, rat and mouse), but differed in having higher proportions of glutamic acid, alanine and isoleucine. Amino-terminal amino acid sequence analysis up to the 47th residue showed close homology between calf apo-AI and those of the mammals with which it was compared. However, residues 2, 7, 20 and 22 in calf AI (i.e. aspartic acid, serine, glutamic acid and isoleucine, respectively) were substituted by glutamic acid, proline or glutamine, aspartic acid, and valine or leucine respectively, in the other mammals.

Seasonal variations of plasma lipids and lipoproteins in the hedgehog, an animal model for lipoprotein (a) metabolism: relation to plasma thyroxine and testosterone levels.

We describe a study of the seasonal variations of hedgehog plasma lipids and lipoproteins and their correlation with changes in the activities of the thyroid and testis. In ten male hedgehogs, plasma concentrations of lipids, thyroxine and testosterone were assayed each month for 1 year beginning in September, while plasma lipoproteins from five of these animals were analyzed at the same dates using density gradient ultracentrifugation. All classes of plasma lipids (cholesterol, total glycerol and phospholipids) exhibited statistically significant seasonal variations in their respective concentrations, with simultaneous maxima (cholesterol: 207 +/- 39 mg/100 ml; total glycerol: 50 +/- 9 mg/100 ml; phospholipids: 266 +/- 25 mg/100 ml) during late fall-early winter, i.e., during the period of the year when plasma levels of both thyroxine and testosterone were minimal. Plasma lipids subsequently decreased to minimal levels either in early summer (cholesterol: 129 +/- 18 mg/100 ml; phospholipids: 178 +/- 20 mg/100 ml) or in late winter (total glycerol: 22 +/- 9 mg/100 ml). Very low density lipoproteins (d less than 1.015 g/ml) were found at low levels (less than 15 mg/100 ml) during the cold months, and then became detectable as trace components only. The total concentration of the mixed lipoprotein population (i.e., low density lipoproteins, Lp(a), and high density lipoprotein (HDL)-like particles) in the d 1.015-1.065 g/ml interval decreased by almost 50% from January to February (from 164.3 to 89.2 mg/100 ml), i.e., following a 10-fold increase in the level of plasma testosterone, and immediately before the rapid doubling in plasma thyroxine concentration. The staining intensity of the electrophoretic band with migration characteristics corresponding to those of Lp(a) decreased considerably during winter. At the same period of the year, lower density (1.032-1.055 g/ml) HDL-like particles disappeared. The concentration of lipoproteins with d 1.065-1.162 g/ml, which included Lp(a) particles in addition to typical HDL, equally underwent seasonal variations. These variations consisted of two successive maxima in late fall (426.4 mg/100 ml) and late winter (458.3 mg/100 ml) with two subsequent decreases leading to minima in February (327.8 mg/100 ml) and August (257.1 mg/100 ml). Finally, very high density lipoproteins (d 1.162-1.259 g/ml) were heterogeneous, containing both cholesterol-rich (d 1.162-1.227 g/ml) and phospholipid-rich (d 1.194-1.259 g/ml) subpopulations.(ABSTRACT TRUNCATED AT 400 WORDS)

[Detection of visna-maedi virus antibodies and antigens by immunoblotting]. / Détection d'anticorps et d'antigènes du virus de Visna-Maedi par immunotransfert.

The visna-maedi virus was immunologically diagnosed using an immunoblotting technique from an antigenic preparation of the visna-maedi virus K796 purified by sucrose density gradient centrifugation. After SDS-PAGE electrophoresis and transfer onto a nitrocellulose sheet, the immunoblotting procedure was adapted to the search for specific antibodies in ovine serum samples. The results obtained showed that, in the natural visna-maedi disease, antibodies are not systematically detectable against every viral protein of the virus core. We have demonstrated the existence of antibodies directed against the proteins coded by the gag and the pol genes.

Lipoprotein[a] is the major apoB-containing lipoprotein in the plasma of a hibernator, the hedgehog (Erinaceus europaeus).

We have undertaken studies aimed at elucidating the interrelationships existing between the seasonal modifications in endocrine status (already demonstrated by Saboureau, M., and J. Boissin. 1978. C.R. Acad. Sci. (Paris) 286D: 1479-1482) and plasma lipoprotein metabolism in the male hedgehog. During the course of these studies, we discovered that a lipoprotein comparable to human Lp[a] was a prominent component of the plasma lipoprotein spectrum in the hedgehog. This lipoprotein was present in the 1.040-1.100 g/ml density range (approximately), exhibited pre beta mobility upon agarose gel electrophoresis, and its Stokes diameter was 275 A. Its apolipoprotein moiety consisted of two proteins with molecular weights and amino acid compositions similar to those of human apoB-100 and apo[a], respectively. These two apolipoproteins were present in hedgehog Lp[a] as a complex that could be dissociated using dithiothreitol and whose stoichiometry could be 1:1. Lp[a] polymorphism due to size heterogeneity of apo[a] appeared to be present in the hedgehog as in man. The chemical composition of hedgehog Lp[a], obtained from animals bled during spring and summer, differed from that of its human counterpart in that the proportion of triglycerides was approximately three times higher in the hedgehog particle (13% vs. 4%), to the detriment of cholesteryl esters. Dissociation of the apoB:apo[a] complex has allowed us to obtain Lp[a] devoid of its specific polypeptide (Lp[a-]), a particle that retained the characteristics of Lp[a] as regards its lipid composition but whose Stokes diameter decreased by 30 to 40 A. The plasma concentration of LDL particles, defined as lipoproteins containing apoB-100 as their sole apolipoprotein constituent, was considerably lower than that of Lp[a]. These findings suggest that the hedgehog could be a unique animal model for studies regarding Lp[a] metabolism.

Subfractionation of 1.006-1.063 g/ml components of badger plasma lipoproteins by using heparin-Sepharose affinity chromatography: relevance to the endocrine regulation of lipoprotein metabolism.

Badger plasma lipoproteins with density 1.006-1.063 g/ml have been subfractionated by means of affinity chromatography on a heparin-Sepharose column, using a modification of the method reported by Weisgraber and Mahley (1980. J. Lipid Res. 21: 316-325). These experiments have provided evidence for the presence of three lipoprotein subfractions hereinafter termed fractions I, II, and III. Fraction I was cholesteryl ester- and phospholipid-rich (ca. 35% and 30% of lipoprotein mass, respectively), and contained apoA-I as its prominent apolipoprotein constituent. In contrast, triglyceride-rich fractions II and III both exhibited a complex apolipoprotein pattern, including apoB-100, apoA-I, and apoE whose amino acid composition and NH2-terminal sequence in the badger are reported. However, fraction III appeared markedly enriched in apoE when compared to fraction II. On polyacrylamide gel electrophoresis, fraction I presented as a spectrum of particles with diameters in the 140-190 A range. In contrast, fraction II migrated as a single band with a diameter of approximately 200 A, and fraction III presented as a single band or a doublet with a diameter of 195-200 A. The respective plasma concentrations and chemical compositions of the three chromatographic fractions were determined at four different dates of the year (i.e., April, August, November, and January), each of which corresponded to a different endocrine status in the badger. Thus hypothyroidism appeared to be associated with an increase in the concentration of fraction I, while the lowering in summer of the plasma level of testosterone correlated well with an increase in the concentration of fraction II. At the same time, the respective proportions of hydrophobic lipids in this latter material modified with an increase of triglycerides. Finally, both the apolipoprotein pattern of fraction III, and the chronologic profile of the successive variations of its concentration, suggest that it could represent a metabolic precursor of fraction II. These results suggest that the respective metabolism of the lipoproteins constituting the three chromatographic fractions could be under control by thyroid and testis secretions, operating via a complex combined regulation of the activities of the enzymes and receptors involved in these metabolic processes.

A year-long study of changes induced by castration in the plasma lipid and lipoprotein spectrum in the European badger.

In man, an influence of male sex hormones on plasma lipid transport is well established; however, recent data on this subject in the literature are both relatively lacking and occasionally conflicting. The male European badger exhibits seasonal variations of large amplitude in its gonadic function. We have therefore attempted to establish the influence of male sex steroids on plasma lipids and lipoproteins in this species. For this purpose, we have examined the plasma lipid and lipoprotein spectrum in a group of castrated male badgers every month for a year, non-operated animals being used as controls. Our analyses included measurement of plasma lipid levels, density gradient ultracentrifugation of lipoproteins, electrophoresis of lipoproteins and apolipoproteins, and evaluation of plasma testosterone and thyroxine levels. The differences observed between the 2 groups of animals were maximal during the months when plasma testosterone was elevated in intact badgers (January to July). For this period, castration resulted in higher plasma concentrations of cholesterol, phospholipids and triglycerides, while the latter alone remained significantly more elevated in operated animals until the end of our experiments. With regard to lipoproteins, the main effect of castration consisted of a large augmentation in the concentration of lipoproteins with d approximately equal to 1.027-1.065 g/ml which were responsible for the transport of most of the increased amounts of triglycerides present in the plasma of castrated badgers. The proportion of apoprotein B in the protein moiety of these lipoprotein components was enhanced after castration. Other changes in the lipoprotein spectrum included (1) a moderate increase in the concentration of lipoproteins with d less than 1.015 g/ml and 1.019-1.027 g/ml, and (2) a modification of the respective proportions of high density lipoproteins with d 1.065-1.100 g/ml and d 1.100-1.162 g/ml. Finally, no considerable differences between the 2 groups of animals were noted in the respective percentages of the various chemical constituents in each lipoprotein subfraction assayed, except for those with d 1.023-1.027 g/ml, which, in castrated badgers, did not exhibit the enrichment in triglycerides usually noted during late winter and spring in intact animals.

Isolation and characterization of the major plasma apolipoproteins, A-1 and B, in the European badger, Meles meles.

The two major apolipoproteins of badger serum, apoA-I and apoB, have been isolated and characterized. Apolipoprotein A-I was the principal protein of badger lipoproteins with density 1.063-1.21 g/ml and, in addition, was present in the lipoprotein class with density 1.006-1.063 g/ml. This apolipoprotein displayed an Mr of approximately equal to 27,000-28,000 and was polymorphic (three prominent isoproteins) on isoelectric focusing, with pI values in the range 5.38-5.55. The amino acid profile of badger apoA-I generally resembled those reported in the literature for similar proteins in dog and man. Amino terminal sequence analysis up to the 40th residue showed close homology between the badger, dog, and human proteins; badger and dog apoA-I differed only at residue 24, at which serine in the dog was substituted by glycine in the badger. Several forms of apolipoprotein B were present in badger lipoproteins with densities less than 1.063 g/ml, their distribution and apparent Mr being unaffected by the presence or absence of 1 mM PMSF during the isolation process. The components of higher Mr were essentially represented by a protein with Mr approximately equal to 530,000-550,000 (apoBH) as determined by SDS-polyacrylamide electrophoresis; this protein predominated both in lipoproteins with d 1.006-1.063 g/ml and in those with d less than 1.006 g/ml. In addition, proteins with approximate Mr values of 490,000, 450,000, and 190,000, respectively, were present as minor components. A lower Mr form (250,000, apoBL), was observed only in lipoproteins with d less than 1.006 g/ml.(ABSTRACT TRUNCATED AT 250 WORDS)

A year-long study of changes induced by thyroidectomy in the plasma lipid and lipoprotein spectrum in the European badger.

Hypothyroidism is associated with hypercholesterolemia and increased risk for atherosclerotic disease. The European badger exhibits large seasonal changes in thyroid activity and the annual minimum of plasma thyroxine level in this species occurs at the same period of the year (i.e. late fall) as a pronounced hypercholesterolemia. We examined the plasma lipid and lipoprotein spectrum in a group of thyroidectomized male badgers every month for a year. Non-operated animals were used as controls. Our analyses included measurement of plasma lipid levels, density gradient ultracentrifugation of lipoproteins, electrophoresis of lipoproteins and apolipoproteins, and histological studies. Maximal differences between the two groups of animals were observed during spring, occurring concomitantly with the annual maximum of plasma thyroxine concentration in control badgers. Comparison with the latter animals revealed a permanent hypercholesterolemia and hyperphospholipidemia in thyroidectomized badgers, while their lipoprotein spectrum was characterized by the continual presence of elevated concentrations of cholesterol-rich lipoproteins of d congruent to 1.015 - 1.027 g/ml. The ratio of triglyceride/cholesteryl ester content in such lipoproteins remained constant throughout the year, resembling that noted in intact animals during late fall. Other features distinguishing the lipoprotein spectrum in thyroidectomized badgers were: (1) higher levels of lipoproteins with d 1.027 - 1.065 g/ml and d 1.065 - 1.100 g/ml, and (2) a cholesteryl ester enrichment of both these lipoprotein subclasses. The two groups of animals shared a heterogeneity of low density lipoprotein subfractions isolated on density gradients, together with the presence of apolipoproteins with molecular weights respectively typical of human apolipoproteins A-I and B throughout the low density range. Arterial walls and heart tissues from intact and thyroidectomized animals were free of atherosclerotic lesions at the end of the experimental period.

Distribution and partial characterization of the plasma lipoproteins in the hedgehog (Erinaceus europaeus L.), a hibernator with potential use in the study of the hormonal regulation of lipoprotein metabolism.

The hedgehog is a hibernator in which yearly cycles of several endocrine activities and seasonal variations of plasma lipids have already been demonstrated. We have consequently undertaken a study of plasma lipids and lipoproteins in this animal, bled during late April and May. Plasma cholesterol levels (178 +/- 30 mg/100 ml) were comparable with those in normal humans, while triacylglycerol was lower (46 +/- 17 mg/100 ml) and phospholipids higher (252 +/- 35 mg/100 ml). The main characteristics of the plasma lipoprotein spectrum, as determined by sequential and density gradient preparative ultracentrifugation, analytical ultracentrifugation and gel filtration chromatography, were (1) a low concentration of very-low-density components (d less than 1.006 g/ml, about 20 mg/100 ml); (2) a continuity between the low (1.006-1.063 g/ml) and high (1.063-1.21 g/ml) density components, the former (about 150 mg/100 ml) exhibiting a considerable heterogeneity upon polyacrylamide gel electrophoresis while the latter were largely predominating (570 mg/100 ml); (3) the presence, at a density of 1.087 g/ml, of a band migrating electrophoretically like human low-density lipoproteins, a finding consistent with the results of apolipoprotein electrophoresis, which showed the presence of a high-molecular-weight counterpart to apolipoprotein B, in both the low- and high-density ranges defined above; (4) the presence, throughout the entire density spectrum, of an apolipoprotein with molecular weight and mobility in polyacrylamide/urea gels similar to human apolipoprotein A-I, and (5) the presence of very-high-density lipoproteins (d 1.178-1.259 g/ml) responsible for the transport of approximately 15% of plasma cholesterol and 20% of phospholipids.

Diet-induced and physiologically occurring hypercholesterolemias in the spontaneous hypothyroid European badger (Meles meles L.): a density gradient study of lipoprotein profile.

As previously shown in this laboratory (Laplaud, P. M. et al. J. Lipid Res. 1980. 21: 724-738), the European badger is, with regard to its plasma lipid transport system, an original and complex animal of great potential interest to lipoprotein research. In an effort to study the response of this animal to cholesterol feeding, we gave a diet supplemented with 1% cholesterol to six male badgers (group H) during the late fall period when spontaneous hypercholesterolemia and hypothyroidism occur. Six more male animals of similar age received the standard diet (group C) and were simultaneously used as controls. Plasma lipids were measured using enzymatic methodologies, while the use of a recently described density gradient ultracentrifugation technique allowed detailed examination of lipoprotein composition and polyacrylamide gel electrophoresis of lipoproteins and tetramethylurea-soluble apoproteins in the fractions. The results suggest the superimposition, in H badgers, of the spontaneous and diet-induced hypercholesterolemias, maximum levels being reached in December in both C and H groups. While the two groups were very similar at the beginning of the experiment, highly significant differences (P < 0.01) were subsequently observed between C and H animals in plasma cholesterol and phospholipid concentrations. Density gradient ultracentrifugation provided evidence for the following diet-induced changes in lipoprotein profile: 1) a twofold increase in cholesteryl esters in particles of d < 1.006 g/ml; 2) the occurrence of large amounts of supplementary cholesterol-rich low density lipoproteins, mainly in the 1.019-1.027 g/ml region; 3) an increase in the 1.039-1.055 g/ml low density lipoproteins; and 4) a change in the ratio of the concentrations of high density lipoproteins of d 1.065-1.100 g/ml and d 1.100-1.162 g/ml, to the benefit of the former. Electrophoresis of the density gradient fractions revealed marked heterogeneity, especially in the low density part of the spectrum. Electrophoresis of the low molecular weight, tetramethylurea-soluble apoproteins failed to show marked differences between C and H badgers. However, chromatographic determination of the proportion of apoB in the protein moiety of the two main low density components showed that 1) it was consistently low, 2) its contribution to the higher density fraction (d 1.039-1.046 g/ml) was unaffected by the hypercholesterolemic diet (being about 25% in both C and H animals), and 3) its contribution to the lower density fraction (d 1.019-1.027 g/ml) decreased under the same nutritional conditions, representing about 20% in C as compared to about 10% in H badgers.-Laplaud, P. M., Beaubatie, and D. Maurel. Dietinduced and physiologically occurring hypercholesterolemias in the spontaneous hypothyroid European badger (Meles meles L.): a density gradient study of lipoprotein profile.

Further characterization of the changes occurring in the plasma lipoprotein spectrum in the European badger (Meles meles L.) during winter.

The plasma lipoprotein pattern in the European badger has been shown previously to undergo marked and complex quantitative and qualitative seasonal modifications (Laplaud, P.M. et al., 1980, J. Lipid Res., 21, 724-738). However, the conventional ultracentrifugal techniques then in use in our laboratory were of insufficient discriminating power with regard to the numerous lipoprotein fractions whose presence was suggested by our analyses. In the present study, a new density gradient ultracentrifugation procedure was applied to the more detailed determination of the distribution of plasma lipoproteins. The first series of analyses was performed in early December and the second in March, i.e. at the dates when the maximum and minimum, respectively, of lipidemia occur in this species. The fractions thus obtained, each of which corresponded to a narrow density interval, were analyzed subsequently for chemical composition, appearance upon polyacrylamide gel electrophoresis, and for their content of tetramethylurea-soluble apolipoproteins in alkaline-urea gels. Changes occurring from December to March included a large decrease in the plasma concentration of the 1.015-1.065 g/ml lipoproteins, chemical analysis of this material being compatible with the presence of at least two lipoprotein populations. On the other hand, high-density lipoproteins (1.065-1.162 g/ml) appeared less variable in chemical composition, although the proportion of those with lower density decreased considerably in early spring. Polyacrylamide gel electrophoresis of the native fractions showed multiple bands in most of them; the tetramethylurea-soluble apoprotein profile remained similar at the two dates considered with an apolipoprotein A-I-like component present in large amounts throughout the entire low- and high-density ranges.

A spontaneously seasonal hypercholesterolemic animal: plasma lipids and lipoproteins in the European badger (Meles meles L.).

The European badger has previously been shown to exhibit yearly cycles of locomotor activity, endocrine secretions, and body weight, as well as seasonal variations in plasma cholesterol. Over a period of 2 years, we have followed the plasma levels of free and esterified cholesterol, triglycerides and phospholipids, and of plasma lipoproteins (by means of polyacrylamide gel electrophoresis, agarose column chromatography and preparative and analytical ultracentrifugation). Some preliminary observations on the qualitative characteristics of the plasma apoproteins, obtained by application of electrophoretic techniques, are also described. Our results provide evidence for considerable synchronous and spontaneous variations of each of the plasma lipid components studied, all of them reaching a maximum in late autumn/early winter, then decreasing to a minimum in early spring. In some animals, the amplitude of observed variations was as large as 650% for total cholesterol, 420% for phospholipids and 180% for triglycerides. While the plasma concentration of very low density lipoproteins (d < 1.006 g/ml) remained at low or moderate levels, major changes in the lipoprotein spectrum occurred in the low density (1.006--1.063 g/ml) and high density (1.063--1.21 g/ml) lipoproteins, these two classes exhibiting marked heterogeneity. This led to an autumn/winter prominence of the 1,006--1.063 g/ml components and of those in the lower part of the high density range, with an enrichment in cholesterol in lipoproteins in the low density region. These phenomena occur simultaneously and/or immediately after the annual minimum of plasma thyroxine concentration in the species considered. In contrast, early spring patterns displayed more classical features with higher density lipoproteins predominating. Our findings thus suggest that the badger may provide a useful model for future experiments regarding the hormonal regulation of plasma lipid transport as well as the metabolism and physiopathological implications of some cholesterol-rich lipoproteins.