RESUMO
Tangier disease (TD) is characterized by severe high-density lipoproteins (HDL) deficiency, hypercatabolism of HDL constituents, impaired cellular cholesterol efflux, and mutations in the gene of ATP-binding cassette 1 (ABC-1). In the present study, we determined plasma lipid and apolipoprotein levels, and HDL subpopulations, in 110 subjects from a large TD kindred in which the proband was homozygous for an A-->C missense mutation at nucleotide 5338 of the ABC-1 transcript. In the proband HDL-C, apoA-I, and apoA-II concentrations were 2, 1, and 2 mg/dl, respectively, apoA-I was present only in prebeta(1), while apoA-II was found free of apoA-I in two distinct alpha mobility subpopulations with different sizes. The smaller size particles contained only apoA-II while the larger one contained apoA-II and apo(a). Relative to unaffected male relatives (n=30), male heterozygotes (n=21) had significant reductions (P<0.001) in plasma HDL-C (-45%), apoA-I (-34%), apoA-II (-59%), apoA-IV (-40%), Lp(a) (-62%), and apoB (-55%) concentrations, and a significant increase (P<0.05, +33%) in plasma apoC-III levels. Female heterozygotes (n=11) similarly had significant reductions (P<0.001) in the concentrations of plasma HDL-C (-42%), apoA-I (-27%), apoA-II (-52%), Lp(a) (-27%), and (P<0.01) apoA-IV (-28%), apoB (-13%), and a significant increase (P<0.05) in plasma apoE levels (+29%) as compared to unaffected female relatives (n=41). Large size HDL subpopulations, especially the two LpA-I particles: alpha(1) and prealpha(1) were dramatically reduced in both male and female heterozygotes relative to their unaffected family members. Since apoA-II decreased more than apoA-I in both male and female heterozygotes, the ratios of apoA-I/apoA-II were significantly (P<0.01) increased. The prevalence of CHD was 60% higher in the 32 heterozygotes than in the 71 unaffected relatives even though the latter group was on average 7 years older. We conclude that TD homozygotes have only prebeta(1) apoA-I-containing HDL subpopulations, while heterozygotes have HDL that is selectively depleted in the large alpha(1), prealpha(1), and alpha(2), prealpha(2) subpopulations, resulting in HDL particles that are small in size, poor in cholesterol, but relatively enriched in apoA-I compared to those of their unaffected relatives. These abnormalities appear to result in a higher risk of CHD in heterozygotes than in unaffected controls.
Assuntos
Heterozigoto , Homozigoto , Lipoproteínas HDL/sangue , Lipoproteínas HDL/classificação , Doença de Tangier/sangue , Doença de Tangier/genética , Adulto , Idoso , Apolipoproteínas/sangue , Doença das Coronárias/etiologia , Feminino , Humanos , Lipídeos/sangue , Lipoproteínas HDL/química , Masculino , Pessoa de Meia-Idade , Tamanho da Partícula , Fatores de Risco , Caracteres Sexuais , Doença de Tangier/complicaçõesRESUMO
High density lipoproteins (HDLs) and their subspecies play a role in the development of coronary heart disease (CHD). HDL subpopulations were measured by 2-dimensional nondenaturing gel electrophoresis in 79 male control subjects and 76 male CHD patients to test the hypothesis that greater differences in apolipoprotein (apo)A-I-containing HDL subpopulations would exist between these 2 groups than for traditional lipid levels. In CHD subjects, HDL cholesterol (HDL-C) was lower (-14%, P<0.001), whereas total cholesterol and the low density lipoprotein cholesterol/HDL-C ratio were higher (9% [P:<0.05] and 21% [P:<0.01], respectively) compared with control levels. No significant differences were found for low density lipoprotein cholesterol, triglyceride, and apoA-I levels. In CHD subjects, there were significantly (P:<0.001) lower concentrations of the large lipoprotein (Lp)A-I alpha(1) (-35%), pre-alpha(1) (-50%), pre-alpha(2) (-33%), and pre-alpha(3) (-31%) subpopulations, whereas the concentrations of the small LpA-I/A-II alpha(3) particles were significantly (P:<0.001) higher (20%). Because alpha(1) was decreased more than HDL-C and plasma apoA-I concentrations in CHD subjects, the ratios of HDL-C to alpha(1) and of apoA-I to alpha(1) were significantly (P:<0.001) higher by 36% and 57%, respectively, compared with control values. Subjects with low HDL-C levels (=35 mg/dL) have different distributions of apoA-I-containing HDL subpopulations than do subjects with normal HDL-C levels (>35 mg/dL). Therefore, we stratified participants according to HDL-C concentrations into low and normal groups. The differences in lipid levels between controls and HDL-C-matched cases substantially decreased; however, the significant differences in HDL subspecies remained. Our research findings support the concept that compared with control subjects, CHD patients not only have HDL deficiency but also have a major rearrangement in the HDL subpopulations with significantly lower alpha(1) and pre-alpha(1-3) (LpA-I) and significantly higher alpha(3) (LpA-I/A-II) particles.
Assuntos
Apolipoproteína A-I/metabolismo , Doença das Coronárias/metabolismo , Lipoproteínas HDL/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Análise de Variância , Apolipoproteína A-I/química , Colesterol/sangue , HDL-Colesterol/sangue , LDL-Colesterol/sangue , Doença das Coronárias/sangue , Eletroforese em Gel Bidimensional , Humanos , Lipoproteínas HDL/química , Masculino , Pessoa de Meia-Idade , Fatores de RiscoRESUMO
Heart attacks frequently occur in normolipidemic subjects with low concentration of high density lipoproteins (=35 mg/dL). A low-fat diet is generally recommended to patients with coronary heart disease. A low-fat diet decreases both low density (LDL) and high density lipoproteins (HDL). We have shown that on an Average American Diet, subjects with different HDL-cholesterol (HDL-C) levels have different HDL subpopulation profiles. In low HDL-C subjects (=35 mg/dL), the apoA-I-only subpopulation alpha(1) is significantly decreased compared to individuals with normal HDL-C levels (>35 mg/dL). We hypothesized that as subjects with low HDL-C already have low HDL concentrations, the major decrease of HDL-C will occur in subjects with normal HDL-C when a low-fat diet is consumed. Normolipidemic male subjects consumed three diets differing in total fat and saturated fat composition (AAD: 37%, Step-1: 28%, Step-2: 24% total fat) for 6 weeks in a three-period double-blind randomized crossover design. Plasma lipids and apolipoproteins were determined and changes in distribution of HDL subpopulations were evaluated. As a result of a low-fat diet, low HDL-C individuals slightly decreased their HDL-C, but substantially decreased their LDL-C resulting in a significant improvement in the LDL-C/HDL-C ratio. However, subjects with normal HDL-C levels decreased both their LDL-C and HDL-C resulting in an unchanged LDL-C/HDL-C ratio. We also observed significant differences in response to low-fat diets in HDL-C and alpha(1) concentrations between low and normal HDL-C subjects. In the normal HDL-C group, consumption of a low-fat diet also resulted in redistribution of apoA-I-containing HDL subpopulations, indicated by a decrease in the large apoA-I-only alpha(1) subpopulation. These data demonstrate that male subjects with low HDL-C respond to a low-fat diet differently than individuals with normal HDL-C.
Assuntos
HDL-Colesterol/sangue , LDL-Colesterol/sangue , Gorduras na Dieta/administração & dosagem , Adulto , Idoso , Apolipoproteína A-I/sangue , Estudos de Casos e Controles , Estudos Cross-Over , Método Duplo-Cego , Humanos , Pessoa de Meia-Idade , PlacebosRESUMO
BACKGROUND: Little information is available about HDL subpopulations during dietary changes. OBJECTIVE: The objective was to investigate the effect of reductions in total and saturated fat intakes on HDL subpopulations. DESIGN: Multiracial, young and elderly men and women (n = 103) participating in the double-blind, randomized DELTA (Dietary Effects on Lipoproteins and Thrombogenic Activities) Study consumed 3 different diets, each for 8 wk: an average American diet (AAD: 34.3% total fat,15.0% saturated fat), the American Heart Association Step I diet (28.6% total fat, 9.0% saturated fat), and a diet low in saturated fat (25.3% total fat, 6.1% saturated fat). RESULTS: HDL(2)-cholesterol concentrations, by differential precipitation, decreased (P < 0.001) in a stepwise fashion after the reduction of total and saturated fat: 0.58 +/- 0.21, 0.53 +/- 0.19, and 0.48 +/- 0.18 mmol/L with the AAD, Step I, and low-fat diets, respectively. HDL(3) cholesterol decreased (P < 0.01) less: 0.76 +/- 0.13, 0.73 +/- 0.12, and 0.72 +/- 0.11 mmol/L with the AAD, Step I, and low-fat diets, respectively. As measured by nondenaturing gradient gel electrophoresis, the larger-size HDL(2b) subpopulation decreased with the reduction in dietary fat, and a corresponding relative increase was seen for the smaller-sized HDL(3a, 3b), and (3c) subpopulations (P < 0.01). HDL(2)-cholesterol concentrations correlated negatively with serum triacylglycerol concentrations on all 3 diets: r = -0.46, -0.37, and -0.45 with the AAD, Step I, and low-fat diets, respectively (P < 0.0001). A similar negative correlation was seen for HDL(2b), whereas HDL(3a, 3b), and (3c) correlated positively with triacylglycerol concentrations. Diet-induced changes in serum triacylglycerol were negatively correlated with changes in HDL(2) and HDL(2b) cholesterol. CONCLUSIONS: A reduction in dietary total and saturated fat decreased both large (HDL(2) and HDL(2b)) and small, dense HDL subpopulations, although decreases in HDL(2) and HDL(2b) were most pronounced.
Assuntos
Doenças Cardiovasculares/prevenção & controle , HDL-Colesterol/sangue , Dieta com Restrição de Gorduras , Gorduras na Dieta/administração & dosagem , Adulto , Fatores Etários , Idoso , População Negra , HDL-Colesterol/classificação , HDL-Colesterol/efeitos dos fármacos , Estudos Cross-Over , Método Duplo-Cego , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fatores Sexuais , Triglicerídeos/sangue , Estados Unidos , População BrancaRESUMO
We compared serum lipid and apolipoprotein predictors of atherosclerosis in cases from the multicenter study, Pathobiological Determinants of Atherosclerosis in Youth (PDAY). The lipid measures included HDL cholesterol (HDL-C) and non-HDL-C, and the apolipoprotein measures included concentrations of apoA1, apoB, and Lp(a), and sizes of the apo(a) proteins. We tested whether the apolipoprotein measures predicted atherosclerotic lesions as well as the more traditional lipid measures. We estimated extent of lesions as fatty streaks or raised lesions (fibrous plaques, complicated or calcified lesions) in 3 sites: thoracic aorta, abdominal aorta, and right coronary artery. Neither apoA1 nor apoB measures were as strongly or consistently correlated with extent of lesions as the corresponding lipid measure (HDL-C and non-HDL-C, respectively). Beyond the basic model that included sex, age, race, smoking status, hypertension, and the lipid measures, apoA1 and apoB added only an average 1.3% increased explanatory ability to the model, whereas HDL-C plus non-HDL-C added an average 2.5%. The results suggest that the traditional lipid measures are more useful than apolipoprotein measures for detecting young persons at high risk of precocious atherosclerosis. Because of large racial differences, the two Lp(a)-related measures, Lp(a) concentrations and apo(a) size, were evaluated in blacks and whites separately. Under these circumstances, neither of the Lp(a)-related measures was strongly or consistently correlated with extent of lesions.
Assuntos
Apolipoproteínas A/análise , Apolipoproteínas B/análise , Arteriosclerose/metabolismo , HDL-Colesterol/análise , Adolescente , Adulto , Distribuição por Idade , Aorta/química , Aorta/patologia , Arteriosclerose/epidemiologia , Arteriosclerose/patologia , Endotélio Vascular/química , Endotélio Vascular/patologia , Feminino , Humanos , Hipertensão/epidemiologia , Hipertensão/metabolismo , Hipertensão/patologia , Lipoproteína(a)/análise , Masculino , Valor Preditivo dos Testes , Fatores de Risco , Distribuição por Sexo , FumarRESUMO
Previously, we have shown that lipid-free apoA-I, when incubated with fibroblasts, will produce lipoproteins of pre-alpha mobility (Asztalos, B. F., et al. 1997. Arterioscler. Thromb. Vasc. Biol. 17: 1630-1636). In order to understand the nature of these pre-alpha particles, we further characterized their lipid content. The pre-alpha particles are high density lipoproteins, having a median density of 1.08 g/ml. They have a surface charge of -18.45 mV. The phospholipid composition of these particles showed that they have 4% each of phosphatidyl ethanolamine and inositol; 69% phosphatidyl choline and 18% sphingomyelin. This phospholipid composition is different from those of plasma HDL (81% phosphatidyl choline, 13% sphingomyelin), plasma membrane on the fibroblasts, and whole fibroblast phospholipid. To demonstrate that the pre-alpha mobility resides in the lipids, lipids from pre-alpha lipoproteins were reconstituted with lipid-free apoA-I. The resultant particles retained their pre-alpha mobility. We conclude that apoA-I may react with specific regions of plasma membrane to acquire this unusual lipid composition and that pre-alpha mobility is caused in part by the unusual phospholipid composition.
Assuntos
Apolipoproteína A-I/metabolismo , Lipoproteínas HDL/metabolismo , Fosfolipídeos/metabolismo , Precursores de Proteínas/metabolismo , Apolipoproteína A-I/química , Sítios de Ligação , Transporte Biológico Ativo , Linhagem Celular , Membrana Celular/metabolismo , Eletroquímica , Humanos , Lipoproteínas HDL/sangue , Lipoproteínas HDL/química , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Fosfolipídeos/química , Precursores de Proteínas/químicaRESUMO
Recent studies have suggested that variations in apoE genotypes may influence the magnitude of plasma lipid changes in response to dietary interventions. We examined the ability of apoE genotype to predict plasma lipid response to reductions in percent of calories from total fat (TF) and saturated fat (SF) in a normolipidemic study population (n = 103) heterogeneous with respect to age, gender, race, and menopausal status. Three diets, an average American diet (34.3% TF, 15.0% SF), an AHA Step 1 diet (28.6% TF, 9.0% SF), and a low saturated fat (Low-Sat) diet (25.3% TF, 6.1% SF) were each fed for a period of 8 weeks in a three-way crossover design. Cholesterol was kept constant at 275 mg/d; monounsaturated and polyunsaturated fat were kept constant at approximately 13% and 6.5% of calories, respectively. Fasting lipid levels were measured during each of the final 4 weeks of each diet period. Participants were grouped by apoE genotype: E2 (E2/2, E2/3, E2/4); E3 (E3/3); E4 (E3/4, E4/4). Relative to the average American diet, both the Step 1 and Low-Sat diets significantly reduced total cholesterol, LDL cholesterol, and HDL cholesterol in all three apoE genotype groups. No evidence of a significant diet by genotype interaction, however, could be identified for any of the measured lipid and lipoprotein end points. Additional analysis of the data within individual population subgroup (men and women, blacks and whites) likewise provided no evidence of a significant diet by genotype interaction. Thus, in a heterogeneous, normolipidemic study population, apoE genotype does not predict the magnitude of lipid response to reductions in dietary saturated fat.
Assuntos
Apolipoproteínas E/genética , Gorduras na Dieta/farmacologia , Ácidos Graxos/farmacologia , Lipídeos/sangue , Adulto , Idoso , Índice de Massa Corporal , Colesterol na Dieta/farmacologia , Estudos de Coortes , Dieta , Carboidratos da Dieta/farmacologia , Ingestão de Energia , Feminino , Frequência do Gene , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo GenéticoRESUMO
This article characterizes products formed by the interaction of purified apolipoprotein (apo) A-I and human fibroblasts. Fibroblasts were incubated with different concentrations of purified apoA-I (1 to 30 micrograms/mL) in tissue culture medium for different periods of time (0 to 24 hours). The medium was then characterized by one- (agarose) and two-dimensional (agarose: polyacrylamide nondenaturing gradient gel) electrophoresis. At any given concentration of apoA-I, the rate of cellular cholesterol efflux appeared linear over 24 hours. Incubating purified apoA-I with fibroblasts for 4 hours, we detected five pre-alpha lipoproteins with particle sizes between 114 and 684 kDa. Formation of pre-alpha lipoproteins was concentration-dependent. At low concentrations (below 5 micrograms/mL apoA-I), all purified apoA-I (with pre-beta mobility) was converted to pre-alpha lipoproteins. At higher concentrations (greater than 5 micrograms/mL apoA-I), more apoA-I remained with pre-beta mobility. The pre-alpha lipoproteins were characterized by colocalization of apoA-I particles with 14C-cholesterol and 32P-phospholipids. Results showed that the pre-alpha particle of lowest molecular weight contained phospholipid and apoA-I but no cholesterol. The remaining pre-alpha particles contained all three substances. When pre-alpha particles were subjected to ultracentrifugation, all particles floated at d < 1.21 g/mL with some of the smallest phospholipid apoA-I only particles being present in the d > 1.21 g/mL fraction. Based on these results, we postulated that in the first stages of reverse cholesterol transport, pre-alpha lipoproteins are formed by the interaction of lipid free apoA-I and peripheral cells.
Assuntos
Apolipoproteína A-I/metabolismo , Colesterol/metabolismo , Lipoproteínas HDL/metabolismo , Fragmentos de Peptídeos/metabolismo , Células Cultivadas , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Fibroblastos/metabolismo , Humanos , Fosfolipídeos/metabolismoRESUMO
Epidemiological studies have established that plasma concentration of HDL is inversely correlated with the risk of coronary heart disease, even in the absence of increased LDL cholesterol levels. We postulate that specific HDL subpopulations may be responsible for antiatherogenic properties of HDL. HDL subpopulations were quantitated by two-dimensional gel electrophoresis in 79 normolipidemic healthy male subjects. To eliminate the influence of diet, volunteers consumed an average American diet for 6 weeks. After the diet period, subjects were stratified according to their HDL cholesterol (HDL-C) levels to low HDL-C < 0.91 mmol/L (< 35 mg/dL), medium > 0.91 < 1.30 mmol/L (> 35 < 50 mg/dL), and high > or = 1.30 mmol/L (> or = 50 mg/dL) groups. Plasma triglycerides and insulin levels were in the normal range, but subjects with low HDL-C levels had higher concentrations of plasma triglycerides and insulin than subjects with medium or high HDL-C concentrations. The absolute concentration (mg/dL) of apoA-I in the largest alpha-migrating HDL subpopulation (alpha 1) was (P < .01) lower in the low HDL-C subjects compared with the medium and high HDL-C groups. The relative concentration (percent distribution) of apoA-I was decreased (P < .01) in alpha 1 and increased (P < .01) in alpha 3 subpopulations. A positive correlation between HDL-C and alpha 1 (P < .001) and a negative correlation between HDL-C and alpha 3 were observed. The inverse correlation of apoA-I distribution (relative concentration) between alpha 1 and alpha 3 suggests an interconversion of alpha 1 and alpha 3 subpopulations, possibly by cholesteryl ester transfer protein. Pre-beta subpopulations showed an inverse trend with HDL-C, while the pre-alpha subpopulation behaved similarly to the alpha-migrating subpopulation. Colocalization of apoA-I and apoA-II particles in the different HDL subpopulations demonstrated that alpha 1, pre-beta 1, and pre-beta 2 subpopulations are apoA-I-only particles rather than apoA-I:A-II particles.
Assuntos
HDL-Colesterol/sangue , Adulto , Idoso , Apolipoproteína A-I/sangue , Doença das Coronárias/etiologia , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
We report the lipid composition of dog plasma and peripheral lymph lipoproteins as separated into pre-beta, alpha, and pre-alpha fractions by agarose gel electrophoresis. Plasma lipoproteins with alpha mobility have a composition different from that of plasma lipoproteins with pre-alpha mobility, having 9% versus 11% free cholesterol, 21% versus 17% cholesterol ester, 1% versus 16% triacylglycerol, and 69% versus 56% phospholipid. On the other hand, lymph alpha and pre-alpha lipoproteins have compositions that are quite similar (9% versus 7% free cholesterol, 17% versus 17% cholesterol ester, 2% versus 4% triacylglycerol, and 71% versus 71% phospholipid). The lipid compositions of plasma and lymph alpha lipoproteins are quite similar (9% versus 9% free cholesterol, 21% versus 17% cholesterol ester, 1% versus 2% triacylglycerol, and 70% versus 72% phospholipid). The lipid compositions of plasma and lymph pre-alpha lipoproteins are different (11% versus 7% free cholesterol, 17% versus 17% cholesterol ester, 16% versus 4% triacylglycerol, and 56% versus 71% phospholipid). Peripheral lymph lipoproteins with pre-beta mobility contained 15% cholesterol, 13% cholesterol ester, 10% triacylglycerol, and 61% phospholipid. Compared with plasma, peripheral lymph lipoproteins are free cholesterol-enriched in all fractions. Calculated stoichiometric ratios of lipid to apoA-I per particle, alpha lipoproteins have two molecules of apoA-I per particle, and pre-alpha lipoproteins have four molecules of apoA-I per particle.
Assuntos
Lipídeos/sangue , Lipoproteínas HDL/sangue , Linfa/metabolismo , Animais , Apolipoproteína A-I/química , Cães , Feminino , Lipídeos/química , Lipoproteínas HDL/química , Estrutura Molecular , Peso MolecularRESUMO
The influence of dilution on apolipoprotein (apo) A-I-containing subpopulations was studied in human plasma. Agarose electrophoresis and two-dimensional agarose nondenaturing gradient polyacrylamide gel electrophoresis were used. Both in one- and two-dimensional electrophoresis, an increase of charge was observed that resulted in an increase of subpopulations with pre-alpha mobility. Dilution of plasma also resulted in a decrease in the size of apo A-I-containing pre-beta 1 subpopulations. The existence of smaller pre-beta 1 particles was confirmed by subjecting undiluted and 8x diluted plasma to 3% to 16% nondenaturing gradient gel electrophoresis for 4 hours. In addition to the generally observed pre-beta 1 subpopulations, smaller particles similar in size to the free apo A-I were detected even in the undiluted plasma. During dilution, the proportion of larger pre-beta 1 particles decreased while the smaller ones increased, and in 8x diluted plasma, almost all the pre-beta 1 was present in smaller sizes. Using 3% to 35% nondenaturing polyacrylamide gels run for 24 hours, no pre-beta 1 particles could be detected in 8x diluted plasma because the small pre-beta 1 electrophoresed out. These studies show that pre-beta 1 particles can be converted to smaller ones during dilution. It also was demonstrated that "free apo A-I-like" pre-beta 1 particles are present in undiluted plasma. The presence of these particles may have important physiological and pathophysiological functions.
Assuntos
Apolipoproteína A-I/metabolismo , Eletroforese em Gel de Ágar , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Lipoproteínas de Alta Densidade Pré-beta , Humanos , Lipoproteínas HDL/sangueRESUMO
Serum amyloid A proteins (SAAs), a family of homologous molecules, are apolipoproteins of high density lipoprotein (HDL). They can be divided into two groups. The first group comprises the well-characterized acute phase SAAs that associate with HDL during inflammation, thereby remodeling the HDL particle by displacing apolipoprotein (apo)A-I. The second group consists of the recently discovered constitutive SAAs, mouse SAA5 and human SAA4. They exist as minor apolipoproteins on HDL but constitute more than 90% of the total SAA during homeostasis. We have characterized human SAA4 as an apolipoprotein. During homeostasis, SAA4 is synthesized only in the liver. Purification of SAA4 has been described and its plasma concentration has been established at 55 +/- 13 micrograms/ml in 26 healthy individuals. It was present on all HDL density classes and very low density lipoprotein (VLDL) but was absent from low density lipoprotein (LDL). Using two-dimensional electrophoresis and phosphorimaging, SAA4 was found to be associated with a specific subpopulation of only three HDL particles, not involved in the initial cholesterol transfer from cells.
Assuntos
Apolipoproteínas/isolamento & purificação , Proteína Amiloide A Sérica/isolamento & purificação , Reação de Fase Aguda/sangue , Apolipoproteínas/biossíntese , Apolipoproteínas/sangue , Humanos , Lipoproteínas HDL/sangue , Lipoproteínas HDL/classificação , Lipoproteínas HDL/isolamento & purificação , Lipoproteínas VLDL/sangue , Lipoproteínas VLDL/classificação , Lipoproteínas VLDL/isolamento & purificação , Testes de Precipitina , Proteína Amiloide A Sérica/biossíntese , Distribuição TecidualRESUMO
Correlation between coronary heart disease and lipoprotein size and composition is well documented. Within the low-density lipoprotein (LDL) family the small LDL particles are associated with increased risk of coronary heart disease. These particles also have increased apolipoprotein (apo) B content. The appearance of these small LDL particles is the manifestation of complex alteration of plasma lipoprotein metabolism. The LDL size is influenced by genetic, endocrine, and environmental factors. Within the high-density lipoprotein (HDL) family the decrease of larger HDL2 particles is associated with coronary heart disease. HDLs can also be separated according to their apoprotein composition into particles containing lipoprotein (Lp)A-I only and particles containing LpA-I and LpA-II. Most studies have shown that the concentration of LpA-I-only particles decreases in coronary heart disease. HDLs are remodeled in the circulation and this remodeling continues in vitro after the blood is taken. Therefore adequate preservation of blood samples is necessary.
Assuntos
Doença da Artéria Coronariana/sangue , Lipoproteínas LDL/sangue , Tamanho da Partícula , Humanos , Lipoproteínas HDL/sangue , Fatores de RiscoRESUMO
Two-dimensional electrophoresis has been used to resolve 12 distinct apo A-I-containing high-density lipoprotein (HDL) subpopulations in human plasma. The subpopulations were quantitated by 125I-labeled, monospecific antibody and phosphor-imaging. Modification and standardization of the agarose electrophoresis (first dimension) enabled us to recognize new HDL subpopulations. Lipoprotein mobilities in agarose were expressed relative to the mobility of the sample's endogenous albumin. We demonstrated the presence of lipoproteins with mobilities faster than and similar to albumin, as well as subpopulations with mobilities slower than albumin. We refer to these as pre alpha, alpha and pre beta, respectively. Lipoprotein molecular sizes were determined with a non-denaturing polyacrylamide gradient gel electrophoresis (PAGE) (2% to 36%) in the second dimension. Internal standard of 125I-labeled proteins of known molecular size was run simultaneously in each gel permitting accurate size determination. We have demonstrated that ultracentrifugally-isolated lipoproteins are different from the native apo A-I-containing subpopulations. The major difference observed was the loss of pre beta 1 and pre beta 2 particles from the d < 1.21 g/ml fractions to the d > 1.21 g/ml fractions. Possible physiologic and pathologic implications of these findings are also discussed.
Assuntos
Apolipoproteína A-I/análise , Eletroforese em Gel Bidimensional/métodos , Lipoproteínas HDL/sangue , Albuminas , Feminino , Humanos , Lipoproteínas HDL/química , Masculino , Peso Molecular , UltracentrifugaçãoRESUMO
To study in vivo reverse cholesterol transport, dog plasma and lymph apo A-I-containing subpopulations were compared by two-dimensional electrophoresis. Charge and size of subpopulations were similar in plasma and lymph, but the distribution of subpopulations varied considerably. An increase in pre-beta and pre-alpha particles in lymph suggests these changes are a reflection of in vivo reverse cholesterol transport.
Assuntos
Apolipoproteína A-I/análise , Eletroforese em Gel Bidimensional/métodos , Espaço Extracelular/química , Lipoproteínas HDL/sangue , Linfa/química , Animais , Transporte Biológico , Colesterol/metabolismo , Cães , Lipoproteínas HDL/química , Lipoproteínas HDL/isolamento & purificaçãoRESUMO
An exponential gradient gel with 0-35% acrylamide and 0.5% agarose was developed for electrophoresis of intact lipoproteins with subsequent electroimmunoblotting. The system resolved in a single gel lipoprotein-associated proteins of sizes from 'free' apoproteins to VLDL. Reproducibility between gels was good (coefficient of variation < 8%). Examination of the effect of mild glutaraldehyde fixation on immunodetection showed variable results (lack of effect on apos (a), AII, and AIV; inhibition of apoB; enhancement of apos AI and E). The composite gel system described here will simplify analysis of apolipoprotein distributions in both health and disease and therefore will likely be useful in future clinical applications.
Assuntos
Apolipoproteínas/isolamento & purificação , Immunoblotting/métodos , Apolipoproteínas/sangue , Eletroforese em Gel de Ágar , Eletroforese em Gel de Poliacrilamida , Humanos , Hiperlipidemia Familiar Combinada/sangue , Lipoproteínas HDL/sangue , Lipoproteínas HDL/isolamento & purificação , Lipoproteínas LDL/sangue , Lipoproteínas LDL/isolamento & purificação , Lipoproteínas VLDL/sangue , Lipoproteínas VLDL/isolamento & purificaçãoRESUMO
In human peripheral interstitial fluid, esterification of cholesterol by lecithin cholesterol acyltransferase (LCAT) was found to occur at a rate of only 10% of that in plasma (5.6 +/- 1.8 compared with 55.6 +/- 7.8 nmol/ml per h). Measurement of cholesterol esterification in the presence of excess reconstituted apoA-I HDL (rA-I HDL) revealed an LCAT activity in interstitial fluid of 24% of that in plasma, indicating that the low rate of esterification could not be caused by limiting mass of LCAT enzyme. When plasma was diluted to the same concentration as in interstitial fluid, the percent cholesterol esterification rate was the same as undiluted plasma and significantly higher than that of interstitial fluid. These findings led us to postulate that poor activation of LCAT in interstitial fluid may result from a change in conformation in apoA-I. To test this hypothesis, a monoclonal antibody AI-11 that inhibits apoA-I activation of LCAT was used to measure apoA-I in interstitial fluid and plasma. Antibody AI-11 recognized interstitial fluid apoA-I poorly, whereas a polyclonal antibody recognized interstitial fluid apoA-I normally. Incubation of antibody AI-11 with high density lipoprotein or rA-I HDL inhibited apoA-I activation of LCAT. We conclude that the altered conformation of apoA-I in interstitial fluid may render it a poor activator of LCAT.
Assuntos
Apolipoproteína A-I/imunologia , Fosfatidilcolina-Esterol O-Aciltransferase/metabolismo , Albuminas/metabolismo , Apolipoproteína A-I/metabolismo , Apolipoproteínas B/metabolismo , Colesterol/metabolismo , Ésteres do Colesterol/metabolismo , Ativação Enzimática , Epitopos , Espaço Extracelular/metabolismo , Humanos , Técnicas In Vitro , Lipoproteínas HDL/metabolismo , Conformação ProteicaRESUMO
In order to better characterize and optimize a typical capture ELISA system for Lp(a) lipoprotein, we have analysed kinetic details of the reaction. Plate coating with polyclonal antibody, recognition of captured analyte with monoclonal antibody, and detection of monoclonal antibody with alkaline phosphatase-labeled antiglobulin were essentially complete after one hour, probably being driven forward by a relative excess of reagent. However, complete capture of the Lp(a) analyte required about 6 h at low input concentrations. Shorter time periods for capture might therefore result in decreased sensitivity and reproducibility. Deviations from linearity in the assay dose response were associated with incomplete capture of Lp(a) and significant depletion of the monoclonal recognition antibody. With the final reaction conditions described, no significant differences in immunochemical reactivity between samples were found by analysis of dose response slopes. Finally, interferences from plasminogen, -20 degrees C storage, anticoagulants, LDL, haemolysis, and bilirubin were minimal.
Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Lipoproteínas/análise , Animais , Lipoproteína(a) , CoelhosRESUMO
Plasma lipoprotein(a), Lp(a), is the most important known genetically controlled independent risk factor for the prediction of early atherosclerosis (AS) and coronary artery disease (CAD) in a significant subpopulation of Caucasians. A sensitive, specific 'capture' enzyme linked immunosorbent assay (ELISA) is reported for the assay of human plasma Lp(a). There is no interference from low density lipoprotein (LDL), plasminogen, or from endogenous lipids, hemoglobin, or bilirubin. An immobilized polyclonal rabbit antibody 'captures' the Lp(a) ligand, and then a monoclonal murine antibody 'recognizes' it. Alkaline phosphatase conjugated rabbit antimouse IgG and para-nitrophenyl phosphate substrate 'detect' and 'indicate' colorimetrically the amount of Lp(a) bound. Quantitation is relative to a commercially available secondary clinical standard. The frequency distribution for a predominantly Caucasian reference population is highly skewed toward the higher concentrations. The median plasma Lp(a) concentration for healthy Caucasians is 80 mg per 1. Relative risk for early myocardial infarction (MI) increases as plasma Lp(a) levels increase above 300 mg per 1. Approximately 20 percent of Caucasians have plasma Lp(a) values above 300 mg per 1. The frequency distributions of plasma Lp(a) in Blacks and Caucasian type II diabetics are different from the healthy Caucasian reference population. The percentiles of Lp(a) values greater than 300 mg per 1 in these latter groups is three times higher. Thorough epidemiologic and clinical studies where groups are segregated by race and ethnic origin are needed for accurate clinical interpretation of plasma Lp(a) results. Only neomycin and niacin are shown to lower plasma Lp(a) levels therapeutically, although anabolic steroid medication causes lower plasma Lp(a) concentrations. Endocrine malfunction also may influence plasma Lp(a) levels.
Assuntos
Ensaio de Imunoadsorção Enzimática , Lipoproteínas/sangue , Animais , Colorimetria , Humanos , Imunoglobulina G/imunologia , Lipoproteína(a) , Lipoproteínas/imunologia , Infarto do Miocárdio/diagnóstico , Coelhos , Valores de Referência , Fatores de RiscoRESUMO
We studied the interstitial fluid concentration of two lipid-metabolizing enzymes (lipoprotein lipase and hepatic triacylglycerol lipase) to determine their importance in interstitial modification of filtered lipoproteins. Despite the use of a very sensitive lipase assay (1 nmol of fatty acid release/ml/hr), lipase activities in plasma and in peripheral and skeletal muscle lymph from control dogs were below the sensitivity of our assay. After heparin injection, hepatic triacylglycerol lipase and lipoprotein lipase activities in plasma were similar. However, the postheparin hepatic triacylglycerol lipase activities in peripheral and skeletal muscle lymph were only 1.4% and 1.1%, respectively, those of plasma. This concentration is considerably less than the lymph concentration of albumin, which has a similar size to the lipases but has a lymph concentration of 30% to 40% of plasma. Lipoprotein lipase activity in peripheral lymph and skeletal muscle lymph was 2.7% and 4.8%, respectively, of plasma activity. Since lipoprotein lipase has a similar size as hepatic triacylglycerol lipase, the disproportionate amount of lipoprotein lipase in lymph as compared to hepatic triacylglycerol lipase could be due to heparin crossing the capillary endothelium and displacing lipoprotein lipase from peripheral cells. Injection of radioactive heparin confirmed that it does cross into the interstitial space in sufficient concentrations to displace lipase from peripheral cells. We conclude that most of the lipase found in lymph after heparin injection is derived from peripheral cells and not from plasma. Furthermore, hepatic triacylglycerol lipase does not play a role in high density lipoprotein remodeling in interstitial fluid. Therefore, it seems likely that the considerable remodeling of high density lipoprotein that we found previously results from its interaction with peripheral cells.