Novel tocotrienols of rice bran suppress cholesterogenesis in hereditary hypercholesterolemic swine.

A tocotrienol-rich fraction (TRF(25)) and novel tocotrienols (d-P(21)-T3 and d-P(25)-T3) of rice bran significantly lowered serum and low density lipoprotein cholesterol levels in chickens. The present study evaluated the effects of novel tocotrienols on lipid metabolism in swine expressing hereditary hypercholesterolemia. Fifteen 4-mo-old genetically hypercholesterolemic swine were divided into five groups (n = 3). Four groups were fed a corn-soybean control diet, supplemented with 50 microg of either TRF(25), gamma-tocotrienol, d-P(21)-T3 or d-P(25)-T3 per g for 6 wk. Group 5 was fed the control diet for 6 wk and served as a control. After 6 wk, serum total cholesterol was reduced 32-38%, low density lipoprotein cholesterol was reduced 35-43%, apolipoprotein B was reduced 20-28%, platelet factor 4 was reduced 12-24%, thromboxane B(2) was reduced 11-18%, glucose was reduced 22-25% (P<0.01), triglycerides were reduced 15-19% and glucagon was reduced 11-17% (P<0.05) in the treatment groups relative to the control. Insulin was 100% greater (P<0.01) in the treatment groups than in the control group. Preliminary data (n = 1) indicated that hepatic activity of the 3-hydroxy-3-methylglutaryl-coenzyme A reductase was lower in the treatment groups, and cholesterol 7alpha-hydroxylase activity was unaffected. Cholesterol and fatty acid levels in various tissues were lower in the treatment groups than in control. After being fed the tocotrienol-supplemented diets, two swine in each group were transferred to the control diet for 10 wk. The lower concentrations of serum lipids in these four treatment groups persisted for 10 wk. This persistent effect may have resulted from the high tocotrienol levels in blood of the treatment groups, suggesting that the conversion of tocotrienols to tocopherols may not be as rapid as was reported in chickens and humans.

Identification of a mutation in the low density lipoprotein receptor gene associated with recessive familial hypercholesterolemia in swine.

Elevated blood plasma cholesterol (hypercholesterolemia) is a major risk factor for coronary artery disease (CAD) in humans. Genetic dissection of polygenic lipid and lipoprotein disorders in swine, a key animal model for the study of familial hypercholesterolemia (FH) and CAD, led to the isolation of a monogenic subphenotype (FH-r), that is inherited in the recessive (r) manner. A genome scan mapped the FH-r locus close to the centromere of chromosome 2. Comparative mapping showed that this region shares homology with a part of human chromosome 19 that harbors the low density lipoprotein receptor (LDLR) locus, and therefore suggested LDLR as the prime candidate gene for FH-r. Cloning and sequencing of hepatic LDLR cDNA from two FH-r/r and one normal (N/N) animals disclosed a single missense mutation (R84C) in a region that corresponds to human exon 4. The C84 mutation cosegregates invariantly with hypercholesterolemia, which strongly suggests that this mutation is responsible for the observed hyperlipidemia.

Genes specifying receptors for F18 fimbriated Escherichia coli, causing oedema disease and postweaning diarrhoea in pigs, map to chromosome 6.

The study comprised 236 pigs selected for resistance or susceptibility to oedema disease. The susceptibility to colonization of the small intestine by an Escherichia coli strain causing oedema disease was determined: (1) by monitoring faecal excretion of weaned pigs orally inoculated with E. coli strain O139:K12(B):H1:F18ab serotype; and (2) by an in vitro adhesion assay using an F18ab positive E. coli strain and small intestinal enterocyte preparations. Susceptibility to adhesion by these bacteria was shown to be controlled by a dominant (B) allele of the ECF18R locus and resistance by the alternative recessive allele (b). Pigs were typed for 14 blood group systems, 11 biochemical polymorphisms and the polymorphism at nucleotide 1843 of the RYR1 locus. Linkage was demonstrated between the locus for F18 E. coli receptors and the loci S, RYR1, GPI, EAH, A1BG and PGD (Z > 20). The most likely gene orders are: S-ECF18R-RYR1-GPI-PGD or GPI-RYR1-ECF18R-S-PGD. The recombination frequencies between ECF18R-S and ECF18R-RYR1 were estimated to be theta = 0.5% and 3.1%, respectively.

Effects of simvastatin on plasma lipids and apolipoproteins in familial hypercholesterolemic swine.

Familial hypercholesterolemia (FHC) in swine, which resembles human familial combined hyperlipidemia, is a complex lipid and lipoprotein disorder associated with the development of severe coronary lesions similar to those occurring in advanced human coronary disease. The disorder is characterized by elevated plasma total cholesterol (TC), triglycerides (TG), LDL-cholesterol (LDL-C), apolipoproteins (apo) B, C-III, and E, and by decreased levels of HDL-cholesterol (HDL-C), apoA-I, and lecithin:cholesterol acyltransferase (LCAT) activity. A dose-response study with simvastatin, a specific inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, was conducted in four treatment groups of FHC animals, exhibiting TC > or = 250 mg/dL. The animals were fed 0, 80, 200, or 400 mg simvastatin daily for 3 weeks. The measured serum parameters included the levels of TC, VLDL-C, LDL-C, HDL-C, TG, lathosterol, apoA-I, B, C-III, and E, as well as LCAT activity. Simvastatin at 200 mg/d significantly decreased the levels of TC (-25%), LDL-C (-27%), lathosterol (-40%), apoB (-22%), apoC-III (-37%), and apoE (-24%) and modestly decreased the levels of HDL-C (-12%) and apoA-I (-11%) (percent relative to the average pretreatment and posttreatment baseline values) but did not affect the levels of TG, VLDL-C, the lathosterol/TC ratio, or LCAT activity. The levels of TC, LDL-C, apoB, and E were also lowered by simvastatin at 80 or 400 mg/d, but to a lesser extent than at 200 mg/d, while the other parameters were not influenced at these doses. The simvastatin-induced decreases of LDL-C, HDL-C, and apoA-I, B, C-III, and E were significantly correlated among each other. These results show that the trend of responses in TC, LDL-C, apoB, apoC-III, and apoE to simvastatin in the FHC swine is similar to that observed in humans, although the drugs is less potent and efficacious in swine, while the results are different from those in humans with regard to the remaining parameters.

Elevated concentrations of plasma lipids and apolipoproteins B, C-III, and E are associated with the progression of coronary artery disease in familial hypercholesterolemic swine.

We reported earlier that a complex familial hypercholesterolemia (c-FHC) phenotype characterized by elevated levels of total plasma cholesterol (TC) and apoB and reduced levels of HDL cholesterol (HDL-C) and apoA-I is associated with the development of spontaneous atherosclerotic lesions in swine. In this study, we investigated concentrations of plasma lipids and apolipoproteins B, C-III, and E in six parental animals of two cholesterol concentration phenotypes and their 32 offspring, which segregated into high, intermediate, and normal cholesterol phenotypes. Subsequently, we compared the extent of atherosclerotic lesion development in coronary arteries to the concentrations of plasma lipids and apolipoproteins in the parents and two offspring per family. Mean concentrations for the high (n = 23), intermediate (n = 13), and normal (n = 2) cholesterol level phenotypes at 4 months of age were TC, 316 +/- 62.2, 159 +/- 17.1, and 105 +/- 12 mg/dL; LDL cholesterol, 275 +/- 63.1, 113 +/- 16.4, and 67 +/- 18.4 mg/dL; HDL-C, 35 +/- 6.1, 41 +/- 5.7, and 33 +/- 6.4 mg/dL; triglycerides, 48 +/- 10.8, 39 +/- 8.0, and 29 +/- 5.7 mg/dL; apoB, 152 +/- 32.5, 80 +/- 7.2, and 48 +/- 5.7 mg/dL; apoC-III, 10 +/- 4.2, 8 +/- 1.7, and 3 +/- 0.1 mg/dL; and apoE, 17 +/- 3.4, 7 +/- 1.7, and 5 +/- 0.7 mg/dL, respectively. Histological analysis of the major coronary arteries from members of the three families showed considerable variation in the severity of lesions, ranging from foci of adaptive intimal thickening consisting of two to six layers of smooth muscle cells to advanced lesions containing necrotic cores, cholesterol clefts, calcification, and hemorrhage (type V). The most extensive lesions occurred only in animals of the high cholesterol phenotype (ie, c-FHC), in which the concentration of TC and apoB progressively increased after 4 months of age, apoC-III, apoE, and triglycerides increased or remained elevated, and HDL-C decreased, except for one animal. Data presented here show that the plasma cholesterol phenotypes in FHC animals are associated with levels of apolipoproteins B, C-III, and E and indicate that the increases in the studied parameters after 4 months of age correlate with the progression of coronary artery disease.

Familial hypercholesterolemia associated with coronary atherosclerosis in swine bearing different alleles for apolipoprotein B.

The development of advanced coronary atherosclerosis was studied in the FHC swine. This model exhibits spontaneous elevations in plasma cholesterol, LDL, and apo B while fed a low-cholesterol, low-fat diet. Hypercholesterolemic animals bearing the apo B genotypes Lpb2/3, 3/3, 3/5, 5/5 and 3/8 developed stenotic coronary lesions containing necrotic cores, fibrous caps, calcification, neovascularization, hemorrhage, and fissuring. Myocardial infarction and myocardial ischemia were also observed. The complicated atherosclerotic plaques observed in this swine model closely resemble advanced coronary artery disease (CAD) found in humans. While coronary atherosclerosis was not observed in the absence of hypercholesterolemia, neither the apo B genotype nor the level of hypercholesterolemia was found to predict the extent of lesion formation. Similar to the case in humans, the familial dyslipidemia associated with the development of CAD in the FHC swine appears to be polygenic.

Identification of new apolipoprotein B epitopes and haplotypes and their distribution in swine populations.

Results from comparative immunogenetic studies on inheritance and identification of four new apolipoprotein B (apoB) allotypes and three additional apoB haplotypes and their distribution in miniature and domestic swine are presented. Immunological surveys on the four new and 16 previously described Lpb allotypes and genetic analysis of their segregation in progenies, of miniature and domestic swine and their crosses, indicate that three new allotypes designated Lpb9, Lpb10 and Lpb101 are individual (mutant) apoB epitopes, each representing a discriminating marker for one of the new apoB haplotypes specified by three new apoB alleles designated Lpb9, Lpb10 and Lpb101. The fourth allotype, Lpb20, is one of the common epitopes forming the alternative epitope pair with Lpb10, and is a constituent of each of the eight previously described and two new apoB haplotypes. The new apoB alleles have so far been found only in miniature swine, with Lpb10 being the most frequent in the Göttingen, Vietnamese Pot-belly and Japanese Miniature, Lpb9 was detected only in Minnesota Miniature and Lpb101 only in Vietnamese Potbelly. The common allotype, Lpb20, shares immunological similarities with human apoB indicating its ancestral origin, whereas none of the alloreagents detecting the three individual apoB variants, Lpb9, Lpb10 or Lpb101, showed cross-reactivity with human apoB, suggesting their exclusive swine origin and evolvement during speciation through mutations.

Familial and diet-induced hypercholesterolemia in swine. Lipid, ApoB, and ApoA-I concentrations and distributions in plasma and lipoprotein subfractions.

Low levels of high-density lipoproteins (HDLs) may constitute an independent risk factor that may be as important as elevated low-density lipoproteins (LDLs) in coronary artery disease (CAD). Concentrations and distributions of lipids, apolipoprotein (apo) B, and apoA-I in the plasma and lipoprotein subfractions of two groups of swine, one with familial hypercholesterolemia (FHC) and the other with diet-induced hypercholesterolemia (DHC), were examined. Normolipidemic (NL) animals served as controls. All pigs carried the Lpb5 apoB mutation, which is known to influence the formation of atherosclerotic lesions. Mean concentrations of serum total cholesterol in NL, DHC, and FHC were 80.0 +/- 9.3, 774.3 +/- 54.5, and 316.5 +/- 36.1 mg/dL, respectively; HDL cholesterol (HDL-C), 33.5 +/- 1.9, 137.0 +/- 9.9, and 22.3 +/- 2.2 mg/dL; triglycerides, 33.0 +/- 16.3, 40.3 +/- 11.7, and 56.8 +/- 7.2 mg/dL; apoB, 35.7 +/- 3.1, 142.0 +/- 4.8, and 169.3 +/- 13.9 mg/dL; and apoA-I, 62.4 +/- 9.3, 170.9 +/- 6.9, and 42.6 +/- 4.8 mg/dL. The distributions of total cholesterol, apoB, and apoA-I in plasma lipoprotein subfractions were also examined. Compared with NL, FHC had fourfold and 4.7-fold increases in total cholesterol and apoB, respectively, distributed in the lower densities (d < 1.043 g/mL), and low HDL-C and apoA-I levels, resulting in a high total cholesterol/HDL-C ratio (14.4:1) and elevated triglyceride levels. DHC was characterized by 10-fold and fourfold increases in total cholesterol and apoB, respectively, resulting in an LDL particle highly enriched in cholesterol, a fourfold increase of HDL-C, an almost threefold increase in apoA-I, and a normal triglyceride level.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies in swine on inheritance and variation in expression of small intestinal receptors mediating adhesion of the K88 enteropathogenic Escherichia coli variants.

Small intestinal enterocyte preparations from 368 pigs were phenotyped by an in vitro adhesion test using six strains of K88 Escherichia (E.) coli, each expressing one of three K88 fimbrial antigenic variants: K88ab, K88ac, or K88ad. All pigs tested were classified into one of four adhesion brush border phenotypes: I (K88ab-, K88ac-, K88ad-); II (K88ab-, K88ac-, K88ad+); III (K88ab+, K88ac+, K88ad-); or IV (K88ab+, K88ac+, K88ad+). The segregation and adhesion affinity data suggest that there are two adhesion affinity receptors for K88ad+ E. coli: a high affinity (adH) and a low affinity (adL) receptor. The high affinity receptor cosegregates with receptors for K88ab and K88ac fimbrial antigens forming together the phenotype IV; the low affinity receptor is associated with the adhesion phenotype II, and its physiological expression is terminated by 16 weeks of age. In contrast, the K88adH receptor is expressed during the entire life cycle. The presence of a mixed adhesion phenotype, K88adM, assumed to be determined by K88ab(-),ac(-),adL(+)/K88ab(+),ac(+),adH(+) heterozygous genotype, is interpreted as an indication that each of the two types of brush border adhesion for the K88ad antigen is expressed on independent enterocytes.

Sequences and expression of the porcine apolipoprotein A-I and C-III mRNAs.

Apolipoprotein A-I (ApoA-I) is the principal protein component of plasma high-density lipoprotein (HDL) and an activator of lecithin:cholesterol acyltransferase. Apolipoprotein C-III (ApoC-III) exchanges between triglyceride-rich lipoproteins and HDL and inhibits the lipolysis and uptake of triglyceride-rich lipoproteins. To study the expression of these Apo-encoding genes in the developing swine, apoA-I and apoC-III cDNAs from a lambda gt11 porcine liver cDNA library and apoC-III from a porcine genomic DNA library were isolated and sequenced. The predicted amino acid (aa) sequence and composition for ApoC-III matched the N-terminal aa sequence and composition of purified swine ApoC-III. Comparison among known ApoA-I and C-III aa sequences from various species revealed strict conservation of amphipathic helices. In adult pigs, the apoA-I mRNA was found predominantly in the intestine and liver, with a small amount detected in the testes. In contrast, apoC-III mRNA was found predominantly in adult liver. Developmentally, hepatic apoA-I and apoC-III mRNAs were expressed in livers of fetal, newborn, and suckling animals. Intestinal apoA-I and apoC-III mRNAs, however, were detected only in postpartum animals. Although intestinal apoA-I mRNA expression continued into the adult, intestinal apoC-III mRNA expression declined sharply after the newborn period.

Decreased lecithin:cholesterol acyltransferase activity in the plasma of hypercholesterolemic pigs.

Lecithin:cholesterol acyltransferase (LCAT) activity levels were determined, as function of plasma total cholesterol (TC) in 13 normocholesterolemic (TC less than 85 mg/dL) and in 28 hypercholesterolemic (TC greater than 98 mg/dL) pigs. The normocholesterolemic group consisted of pigs that carried apo-B allelic genes other than Lpb5 and or Lpb8. The hypercholesterolemic group consisted of Lpb5/x and Lpb5/8 heterozygous and Lpb5/5 homozygous animals. The data reported in this study show that the LCAT activity in the plasma of hypercholesterolemic (HC) pigs (79 +/- 43 units) was significantly lower (p less than 0.0005) compared to the normocholesterolemic controls (175 +/- 45 units). Furthermore, LCAT activity was positively correlated with TC in the normocholesterolemic group (r = +0.54; p less than 0.05), whereas it was negatively correlated with TC in the hypercholesterolemic group (r = -0.73; p less than 0.001). Additional data obtained from incubation experiments suggest that the lower LCAT activity in hypercholesterolemic pigs may be due, at least in part, to inhibition of LCAT activity by components found in the lipoprotein-deficient fractions of the plasma of hypercholesterolemic pigs.

Porcine von Willebrand disease and atherosclerosis. Influence of polymorphism in apolipoprotein B100 genotype.

The relationship of apolipoprotein-B genotype (Lpb) to diet-induced hypercholesterolemia and atherosclerosis was studied in von Willebrand disease (vWD) and normal pigs. Von Willebrand and normal pigs developed comparable levels of hypercholesterolemia (respectively, 757.9 +/- 49.4 versus 772.8 +/- 47.9 mg/dl, P = 0.95). Pigs with Lpb1/5 and Lpb5/8 genotypes, however, developed significantly higher serum cholesterol levels than those with other Lpb genotypes (866.1 +/- 64.0 mg/dl, P = 0.0343). Coronary and aortic atherosclerosis, measured by computer-assisted automated image analyzer, were not significantly different between vWD and normal pigs. Pigs with an Lpb5 allele developed significantly more atherosclerosis than those with the Lpb3/8 or Lpb8/8 genotypes or the rare Lpb1 allele (r greater than or equal to 0.434, P less than or equal to 0.05). Polymorphism in apolipoprotein B100 genotype, then, significantly influenced the severity of diet-induced hypercholesterolemia and atherosclerotic plaque formation in vWD and normal swine without regard to the vWD genotype.

Assignment of the pig apolipoprotein B locus (APOB) to chromosome region 3q24-qter.

The locus for apolipoprotein-B (APOB) has been chromosomally assigned in swine by in situ hybridization of a genomic probe to metaphase chromosomes. As expected based on the observation of extensive linkage conservation and based on the previous assignment of the malate dehydrogenase locus (MDH1) in swine, APOB maps to chromosome 3, specifically to region 3q24-qter. Variations at APOB may represent both in humans and in swine risk factors for hypercholesterolaemia and atherosclerosis. Evidence presented here that the human and porcine APOB occupy evolutionarily conserved chromosome regions provides a basis for using the pig as an animal model to study the APOB associated atherosclerosis risk.

Preferential mammary storage and secretion of immunoglobulin gamma (IgG) subclasses in swine.

Levels of three immunoglobulin gamma (IgG) subclasses, IgGA, IgGB and IgGC, were measured in sow sera, mammary glands, colostrum and milk samples by the single radial immunodiffusion. Serum IgGA and IgGB levels, but not IgGC, showed time dependent variations during gestation and lactation periods. The IgGA level started to decline at day 106 of gestation, reached its minimum at farrowing, and returned to the pre-gestation level 1-3 weeks after weaning. The serum IgGB level started to decrease at day 111 of gestation, reached its minimum at farrowing, and returned to the initial gestation level 1 week after farrowing. A notable decrease (P less than 0.1) in serum IgGC level was observed only on the day of farrowing. IgGA and IgGB were preferentially stored in mammary glands of full-term pregnant sows and secreted into colostrum after farrowing. In contrast, relatively small amounts of IgGC were stored in the mammary glands and secreted into colostrum. These data are interpreted as an indication that the preferential storage of IgGA and IgGB in the mammary gland of sows occurs at the time of significant decreases of these two IgG subclasses in the sera during late gestation and early lactation.

Evidence for linkage between the swine L blood group and the loci specifying the receptors mediating adhesion of K88 Escherichia coli pilus antigens.

Brush borders or enterocytes obtained from the small intestine of 248 pedigreed pigs were tested by adhesion assay in vitro with enterotoxigenic Escherichia (E.) coli strains, each expressing one of the three K88 pilus variants K88ab, K88ac and K88ad. All pigs were classified as belonging to one of the four adhesion phenotypes: I--K88ab(-), ac(-), ad(-); II--K88ab(-), ac(-), ad(+); III--K88ab(+), ac(+), ad(-); and IV--K88ab(+), ac(+), ad(+). Serum or red cells were typed for 15 blood group systems: A-O, B, C, D, E, F, G, H, I, J, K, L, M, N and O; for 11 biochemical polymorphisms: PI1, PI2, PO1A, A1BG, GPI, PGD, TF, HPX, ADA, PGM and AMY; the polymorphism at the IGHG1 locus. Linkage analysis was performed between the alleles at the locus (loci) specifying K88 receptors able to bind one or more different serological types of K88 E. coli and alleles for markers at other loci. Linkage was demonstrated between the locus for the L blood group system and the locus (loci) for K88 E. coli receptors (Z = 3.24), adding one locus (loci) to the previously identified linkage group IV (LGIV) [L-SLB]. The maximum likelihood estimate of the recombination fraction (theta) was 0.23. No evidence was found for linkage between any of the other biochemical and immunogenetic markers and the receptor locus (loci) of K88 E. coli.

Development of complex atherosclerotic lesions in pigs with inherited hyper-LDL cholesterolemia bearing mutant alleles for apolipoprotein B.

The development of atherosclerotic lesions was studied in pigs aged 4 to 54 months with inherited hyperlow-density lipoprotein (LDL) and hypercholesterolemia (IHLC pigs). These pigs bear the Lpb5 and Lpu1 mutant alleles for apolipoproteins B and U and demonstrate spontaneously elevated cholesterol levels, due primarily to elevated LDL. By 1 year of age, IHLC pigs exhibited focal lesions in the major coronary, iliac, and femoral arteries that were composed of macrophage-derived from cells and smooth muscle cells. Peripheral arterial lesions were more fibrous than those found in the coronaries. By 2 years of age, complicated stenotic lesions containing fibrous caps, necrotic cores, cholesterol clefts, granular calcium deposits, and neovascularization deep within the lesion were common in the major coronary vessels. Peripheral vascular lesions were more smooth muscle cell-rich and fibrotic. By 3 years of age, neovascularization was observed throughout the intimal lesion, and hemorrhage and rupture were common. The extent of complicated lesion formation correlated with both the degree and duration of hypercholesterolemia, with the most stenotic lesions observed in the coronary arteries of the oldest animals having the highest cholesterol levels. Thus IHLC pigs with mutant apolipoproteins B and U develop complicated atherosclerotic plaques that closely resemble advanced atherosclerotic lesions found in humans.

Dietary tocotrienols reduce concentrations of plasma cholesterol, apolipoprotein B, thromboxane B2, and platelet factor 4 in pigs with inherited hyperlipidemias.

Normolipemic and genetically hypercholesterolemic pigs of defined lipoprotein genotype were fed a standard diet supplemented with 50 micrograms/g tocotrienol-rich fraction (TRF) isolated from palm oil. Hypercholesterolemic pigs fed the TRF supplement showed a 44% decrease in total serum cholesterol, a 60% decrease in low-density-lipoprotein (LDL)-cholesterol, and significant decreases in levels of apolipoprotein B (26%), thromboxane-B2 (41%), and platelet factor 4 (PF4; 29%). The declines in thromboxane B2 and PF4 suggest that TRF has a marked protective effect on the endothelium and platelet aggregation. The effect of the lipid-lowering diet persisted only in the hypercholesterolemic swine after 8 wk feeding of the control diet. These results support observations from previous studies on lowering plasma cholesterol in animals by tocotrienols, which are naturally occurring compounds in grain and palm oils and may have some effect on lowering plasma cholesterol in humans.

Identification of the ancestral haplotype for apolipoprotein B suggests an African origin of Homo sapiens sapiens and traces their subsequent migration to Europe and the Pacific.

The probable ancestral haplotype for human apolipoprotein B (apoB) has been identified through immunological analysis of chimpanzee and gorilla serum and sequence analysis of their DNA. Moreover, the frequency of this ancestral apoB haplotype among different human populations provides strong support for the African origin of Homo sapiens sapiens and their subsequent migration from Africa to Europe and to the Pacific. The approach used here for the identification of the ancestral human apoB haplotype is likely to be applicable to many other genes.

Concentrations and compositions of plasma lipoprotein subfractions of Lpb5-Lpu1 homozygous and heterozygous swine with hypercholesterolemia.

Pigs with two mutant epitopes, Lpb5 of apolipoprotein B (apoB) and Lpu1 of a yet undefined apolipoprotein, specified by a haplotype Lpb5-Lpu1 and fed a cholesterol-free low fat diet show hypercholesterolemia. The purpose of this study was to establish whether a direct relationship exists between the swine lipoprotein concentration/composition and the genotype for the Lpb5-Lpu1 haplotype; i.e., homozygote versus heterozygote. Lipoproteins of fasted plasma from hypercholesterolemic swine, homozygous (HmHC) and heterozygous (HtHC) for Lpb5-Lpu1, and from normolipidemic (NL) pigs of other Lpb-Lpu haplotypes were separated into five layers by density gradient ultracentrifugation. Layer 1 contained particles of d less than 1.019 g/ml and layer 5 particles of d greater than 1.073 g/ml. Layers 2, 3, and 4 represented subfractions of low density lipoproteins (LDL). The plasma total cholesterol (TC) of the HmHC group (300 +/- 84 mg/dl) was different (P less than 0.05) from the HtHC group (200 +/- 80 mg/dl) and in both HmHC and HtHC, TC was significantly higher (P less than 0.0005 and P less than 0.005, respectively) than that of the NL group (69 +/- 14 mg/dl). The elevation in plasma TC was due to the increased TC in layers 2 and 3: a 13- and 7-fold increase in HmHC and a 7- and 4-fold increase in HtHC in layers 2 and 3, respectively. Parallel increases in unesterified cholesterol were observed in these two layers. Marked increases in apoB were also observed in layers 2 and 3 of HmHC and intermediate increases in apoB in the same two layers of HtHC.(ABSTRACT TRUNCATED AT 250 WORDS)

Separation of swine plasma LDL from Lpb2/3 heterozygotes into two apoB allelic haplotypes, Lpb2 and Lpb3, with apoB epitope specific antibodies.

Studies were performed to investigate the separation of Lpb (lipoprotein B) species present in plasma of heterozygous swine bearing the Lpb2 and Lpb3 apoB mutant genes. Low density lipoprotein (LDL) fractions from Lpb2/2 and Lpb3/3 homozygotes were coupled to a matrix and used to isolate affinity-purified antibodies anti-Lpb2 and anti-Lpb3 from swine alloimmune sera, one with specificity for the Lpb2 epitope(s) and the other for Lpb3. These antibodies in turn were used to construct two immunosorbers, anti-Lpb2 and anti-Lpb3 Sepharose columns. To separate the two Lpb haplotype populations present in LDL, a density gradient ultracentrifuge subfraction (d 1.032-1.043 g/ml) obtained from Lpb2/3 heterozygous pigs was applied to the specific immunosorbers. The retained fraction from the anti-Lpb2 column reacted in the double immunodiffusion test with anti-Lpb2 and anti-Lpb13 immune sera but not with either anti-Lpb3 or anti-Lpb12, while the unretained fractions reacted with anti-Lpb3 and anti-Lpb12 but not with either anti-Lpb2 or anti-Lpb13. The reaction patterns obtained with the two sets of alloimmune sera indicate the existence of two separate lipoprotein populations in LDL: one lipoprotein carrying the Lpb2 and Lpb13 epitopes corresponding to the Lpb2 apoB allele, and the other carrying the Lpb3 and Lpb12 allotypes specified by the Lpb2 gene. Immunoblotting with anti-Lpb2 and anti-Lpb3 and silver staining showed that the epitopes of both isolated LDL subpopulations are associated with apoB-100. Neutral lipid analyses showed no differences between the isolated Lpb2 and Lpb3 lipoprotein species from the Lpb2/3 heterozygotes. These studies demonstrate that plasma LDL subfractions from Lpb heterozygous swine can be separated into two haplotype populations, each corresponding to the product of one apoB gene, and reveal a new insight into the phenotypic expression of plasma LDL, and the LDL phenotype-genotype relationship. Furthermore, this approach will facilitate studies on metabolic differences of two structurally distinct LDL, unaffected by in vitro manipulation, exposed to the metabolic milieu of one individual.