RESUMO
A 56-year-old postmenopausal woman with out-of-hospital cardiac arrest caused by acute myocardial infraction was successfully resuscitated by intensive treatments and recovered without any neurological disability. She was diagnosed as having familial hypercholesterolemia (FH) based on a markedly elevated low-density lipoprotein cholesterol (LDL-C) level and family history of premature coronary artery disease. Genetic testing in her family members showed that a variant of the proprotein convertase subtilisin/kexin type 9 (PCSK9) gene (c.2004Cï¼A, p.S668R), which had been previously reported as having uncertain significance, was associated with FH, indicating that the variant is a potential candidate for the FH phenotype. Next-generation sequencing analysis for the proband also showed that there was a heterozygous mutation of the ATP-binding cassette sub-family G member 5 ( ABCG5) gene (c.1166Gï¼A, R389H), which has been reported to increase LDL-C level and the risk of cardiovascular disease. She was also diagnosed as having type 1 CD36 deficiency based on a lack of myocardial uptake of 123I-labeled 15-(p-iodophenyl)-3-R,S-methyl-pentadecanoic acid in scintigraphy and the absence of CD36 antigen in both monocytes and platelets in flow cytometry. She had a homozygous mutation of the CD36 gene (c.1126-5_1127delTTTAGAT), which occurs in a canonical splice site (acceptor) and is predicted to disrupt or distort the normal gene product. To our knowledge, this is the first report of a heterozygous FH phenotype caused by possibly oligogenic variants of the PCSK9 and ABCG5 genes complicated with type I CD36 deficiency caused by a novel homozygous mutation. Both FH phenotype and CD36 deficiency might have caused extensive atherosclerosis, leading to acute myocardial infarction in the present case.
Assuntos
Hiperlipoproteinemia Tipo II , Infarto do Miocárdio , Membro 5 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Transtornos Plaquetários , Feminino , Doenças Genéticas Inatas , Humanos , Hiperlipoproteinemia Tipo II/complicações , Hiperlipoproteinemia Tipo II/genética , Lipoproteínas/genética , Pessoa de Meia-Idade , Infarto do Miocárdio/complicações , Infarto do Miocárdio/genética , Fenótipo , Pró-Proteína Convertase 9/genética , Receptores de LDL/genéticaRESUMO
BACKGROUND: Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a key regulator of serum low-density lipoprotein (LDL) cholesterol levels. Recently, PCSK9 has additionally been related to metabolic risk factors such as the levels of triglycerides, apolipoprotein B (apoB), insulin, and glucose, as well as body mass index. The purpose of this study was to investigate correlations between serum levels of PCSK9 and apoB-containing atherogenic lipoproteins in patients with coronary artery disease (CAD). METHODS: Serum levels of PCSK9 and lipoprotein(a) [Lp(a)]; small, dense LDL; and oxidized LDL were measured in 101 patients with CAD who were not receiving lipid-lowering therapy. RESULTS: Serum hetero-dimer PCSK9 levels were positively correlated with serum levels of Lp(a) (r = 0.195, p = 0.05); small, dense LDL (r = 0.336, p = 0.0006); and oxidized LDL (r = 0.268, p = 0.008). Multivariate regression analyses showed that serum hetero-dimer PCSK9 was a significant predictor of serum levels of Lp(a) (ß = 0.235, p = 0.01); small, dense LDL (ß = 0.143, p = 0.03); and oxidized LDL (ß = 0.268, p = 0.008). CONCLUSIONS: Serum PCSK9 levels were positively correlated with serum levels of Lp(a); small, dense LDL; and oxidized LDL in patients with CAD. This suggests that the interaction between serum PCSK9 and apoB-containing lipoproteins plays a role in establishing the atherosclerotic status of patients. TRIAL REGISTRATION: UMIN Clinical Trials Registry, UMIN ID: C000000311 .
RESUMO
OBJECTIVE: Sortilin is involved multilaterally in the development of atherosclerosis. Here, we examine the release of soluble sortilin (sSortilin) from platelets and assess the association between circulating levels of sSoritlin and atherothrombosis such as coronary artery disease (CAD). METHODS AND RESULTS: sSortilin levels measured in healthy subjects were higher in serum than in plasma (38.4 ± 8.7 vs. 15.8 ± 2.9 ng/mL; p < 0.0001). Platelets were shown to contain both membrane-bound sortilin and its soluble form lacking the cytoplasmic tail. Stimulation of platelet-rich plasma with collagen induced sSortilin release concomitantly with platelet aggregation, and the release was suppressed by aspirin. In clinical evaluation, plasma sSortilin was detected at significantly higher levels in cardiovascular risk patients with hypertension, dyslipidemia, and/or diabetes without CAD (non-CAD, 18.7 ± 3.3 ng/mL) than in patients with CAD under aspirin therapy (17.1 ± 3.6 ng/mL; p < 0.01) or in healthy controls (16.8 ± 2.9 ng/mL; p < 0.01). In these patients, plasma sSortilin levels were significantly correlated with platelet counts (rs = 0.33; p = 0.0085) and showed significant positive associations with cardiovascular risk factors: low-density lipoprotein cholesterol (rs = 0.37; p = 0.0023), triglycerides (rs = 0.28; p = 0.023), and serum uric acid (rs = 0.30; p = 0.017) in non-CAD, and γ-glutamyltransferase (rs = 0.43; p = 0.020) and high-sensitivity C-reactive protein (rs = 0.33, p = 0.0022) in CAD. CONCLUSION: Elevated plasma sSortilin levels may be associated with in vivo platelet activation and could be a risk factor for atherothrombosis.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/sangue , Proteínas Adaptadoras de Transporte Vesicular/fisiologia , Sistema Cardiovascular/metabolismo , Doença da Artéria Coronariana/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Aspirina/administração & dosagem , Plaquetas/metabolismo , Proteína C-Reativa/metabolismo , Células CHO , Cricetulus , Citoplasma/metabolismo , Feminino , Voluntários Saudáveis , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Plasma/metabolismo , Ativação Plaquetária , Agregação Plaquetária , Proteínas Recombinantes/metabolismo , Fatores de Risco , Adulto JovemRESUMO
AIM: Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a key regulator of serum low-density lipoprotein (LDL) cholesterol levels, and sortilin is linked to lipoprotein metabolism. Although statin therapy increases PCSK9 levels, effects of this therapy on plasma sortilin levels have not been evaluated. The purpose of the present study was to examine the effects of statins on plasma PCSK9 and sortilin levels, and association of statin-induced increase in PCSK9 levels with sortilin. METHODS: Serum lipid levels and plasma PCSK9 and sortilin levels were measured at baseline and 8 months after statin therapy in 90 statin-naive patients with coronary artery disease (CAD). Pitavastatin 4 mg/day was used to treat 44 patients and pravastatin 20 mg/day to treat the remaining 46 patients. RESULTS: For both statin groups, significant increases in hetero-dimer PCSK9 levels (pitavastatin: 31%, pï¼0.0001; pravastatin: 34%, p=0.03) and decreases in sortilin levels (pitavastatin: ï¼8%, p=0.02; pravastatin: ï¼16%, p=0.002) were observed. Although a reduction in LDL cholesterol was greater in the pitavastatin group than in the pravastatin group, no significant differences were observed in percentage changes in hetero-dimer PCSK9 and sortilin levels. A significant positive correlation was observed between percentage changes in hetero-dimer PCSK9 levels and those in sortilin levels (pitavastatin: r=0.359, p=0.02; pravastatin: r=0.276, p=0.06). CONCLUSIONS: Use of pitavastatin and pravastatin increased plasma PCSK9 and decreased sortilin levels. Statin-induced increases in PCSK9 were associated with changes in sortilin in statin-naive patients with CAD.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/sangue , Doença da Artéria Coronariana/tratamento farmacológico , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Pró-Proteína Convertase 9/sangue , Idoso , Doença da Artéria Coronariana/sangue , Feminino , Humanos , Masculino , Estudos Prospectivos , Índice de Gravidade de Doença , Resultado do TratamentoRESUMO
CONTEXT: Proprotein convertase subtilisin/kexin 9 (PCSK9) is known to be a good target to decrease LDL cholesterol (LDL-C) and two forms of PCSK9, mature and furin-cleaved PCSK9, circulate in blood. However, it has not been clarified whether and how the levels of each PCSK9 are affected by LDL-apheresis (LDL-A) treatment, a standard therapy in patients with severe forms of familial hypercholesterolemia (FH). OBJECTIVE: Our objective was to investigate the differences in LDL-A-induced reduction of mature and furin-cleaved PCSK9 between homozygous and heterozygous FH, and between dextran sulfate (DS) cellulose adsorption and double membrane (DM) columns and to clarify the mechanism of their removal. DESIGN: A sandwich ELISA to measure two forms of PCSK9s using monoclonal antibodies was developed. Using the ELISA, PCSK9 levels were quantified before and after LDL-A with DS columns in 7 homozygous and 11 heterozygous FH patients. A crossover study between the two column types was performed. The profiles of PCSK9s were analyzed after fractionation by gel filtration chromatography. Immunoprecipitation of apolipoprotein B (apoB) in FH plasma was performed. RESULTS: Both mature and furin-cleaved PCSK9s were significantly decreased by 55-56% in FH homozygotes after a single LDL-A treatment with DS columns, and by 46-48% or 48-56% in FH heterozygotes after treatment with DS or DM columns. The reduction ratios of LDL-C were strongly correlated with that of PCSK9 in both FH homozygotes and heterozygotes. In addition, more than 80% of plasma PCSK9s were in the apoB-deficient fraction and a significant portion of mature PCSK9 was bound to apoB, as shown by immunoprecipitation. CONCLUSIONS: Both mature and furin-cleaved PCSK9s were removed by LDL-A in homozygous and heterozygous FH either by binding to apoB or by other mechanisms. The ELISA method to measure both forms of plasma PCSK9 would be useful for investigating physiological or pathological roles of PCSK9.
Assuntos
Remoção de Componentes Sanguíneos/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Hiperlipoproteinemia Tipo II/terapia , Pró-Proteína Convertases/sangue , Serina Endopeptidases/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Apolipoproteínas B/sangue , LDL-Colesterol/sangue , Estudos Cross-Over , Feminino , Humanos , Hiperlipoproteinemia Tipo II/sangue , Masculino , Pessoa de Meia-Idade , Pró-Proteína Convertase 9 , Pró-Proteína Convertases/análise , Serina Endopeptidases/análise , Adulto JovemRESUMO
AIM: Apolipoprotein F (apo F), also known as lipid transfer inhibitory protein (LTIP), is a protein component of plasma lipoprotein classes including HDL and functions to inhibit lipid transfer between lipoproteins in vitro. To study the role of plasma apo F, a reliable and sensitive tool for the quantification would be needed. METHODS: We have developed a sandwich ELISA using two monoclonal antibodies for human plasma apo F, and analyzed apo F concentration in 397 Japanese healthy and 221 hypertriglyceridemic subjects. RESULTS: Our ELISA enables apo F to be assayed in the range of 0.6-25 µg/mL with intra- and inter-assay coefficients of variation less than 3.8% and 7.8%, respectively. In healthy subjects, plasma apo F concentration was 12.5±2.9 µg/mL (mean±SD), and was significantly higher in females than in males (p<0.05). By linear regression analysis in healthy subjects, plasma apo F concentration correlated positively with HDL cholesterol and apo A-I levels, and in males but not in females, negatively with apo B and triglyceride levels. It also correlated negatively with intrinsic CETP activity measured using intrinsic apo B-containing lipoprotein as an acceptor, and positively with PLTP mass and apo J levels. Apo F concentration in hypertriglyceridemic patients (10.3±3.1 µg/mL) was lower than in healthy controls (p<0.0001) and correlated positively with PLTP mass. CONCLUSIONS: Our ELISA is reliable and sensitive for the quantification of plasma apo F concentration. This system can be applicable for clinical significance in lipoprotein metabolism and reverse cholesterol transport.
Assuntos
Apolipoproteínas/sangue , Hipertrigliceridemia/sangue , Adulto , Apolipoproteína A-I/sangue , Apolipoproteínas B/sangue , Povo Asiático , Proteínas de Transferência de Ésteres de Colesterol/sangue , HDL-Colesterol/sangue , Clonagem de Organismos , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Japão , Masculino , Proteínas de Transferência de Fosfolipídeos/sangue , Proteínas Recombinantes , Fatores Sexuais , Triglicerídeos/sangueRESUMO
Although much is known about the remodeling of high density lipoproteins (HDLs) in blood, there is no information on that in interstitial fluid, where it might have a major impact on the transport of cholesterol from cells. We incubated plasma and afferent (prenodal) peripheral lymph from 10 healthy men at 37°C in vitro and followed the changes in HDL subclasses by nondenaturing two-dimensional crossed immunoelectrophoresis and size-exclusion chromatography. In plasma, there was always initially a net conversion of small pre-ß-HDLs to cholesteryl ester (CE)-rich α-HDLs. By contrast, in lymph, there was only net production of pre-ß-HDLs from α-HDLs. Endogenous cholesterol esterification rate, cholesteryl ester transfer protein (CETP) concentration, CE transfer activity, phospholipid transfer protein (PLTP) concentration, and phospholipid transfer activity in lymph averaged 5.0, 10.4, 8.2, 25.0, and 82.0% of those in plasma, respectively (all P < 0.02). Lymph PLTP concentration, but not phospholipid transfer activity, was positively correlated with that in plasma (r = +0.63, P = 0.05). Mean PLTP-specific activity was 3.5-fold greater in lymph, reflecting a greater proportion of the high-activity form of PLTP. These findings suggest that cholesterol esterification rate and PLTP specific activity are differentially regulated in the two matrices in accordance with the requirements of reverse cholesterol transport, generating lipid-poor pre-ß-HDLs in the extracellular matrix for cholesterol uptake from neighboring cells and converting pre-ß-HDLs to α-HDLs in plasma for the delivery of cell-derived CEs to the liver.
Assuntos
Apolipoproteína A-I/metabolismo , Líquido Extracelular/metabolismo , Lipoproteínas/metabolismo , Adulto , Humanos , Masculino , Adulto JovemRESUMO
Apolipoprotein (apo) J, clusterin, is ubiquitously expressed in many tissues, and is a component of high-density lipoproteins (HDLs). There is experimental evidence that it may be anti-atherogenic through its effects on cholesterol transport, smooth muscle cell proliferation and lipid peroxidation. HDLs containing apo J and apo A-I carry paraoxonase (PON1), which protects low-density lipoproteins from oxidative modification; however, the extent to which apo J affects coronary heart disease (CHD) is not known. We have developed a sandwich ELISA that enables apo J to be assayed in the range of 13-200 microg/mL. Serum apo J was 52.8+/-0.8 microg/mL (mean+/-SEM; range, 36.0-84.3 microg/mL; n=92) in healthy Japanese men, and 49.3+/-0.5 microg/mL (34.5-72.8; n=241) in healthy Japanese women. Multiple regression of these data and results from 67 men with CHD showed that apo J concentration was unrelated to age, sex or body mass index, but was positively related to serum PON1 (p<0.001) and apo B (p<0.02) concentrations. In women, it was also positively related to blood glucose (p<0.02). After adjusting for its associations with covariates, serum apo J averaged 5.4 microg/mL, lower in CHD men than in controls (p<0.003). Type 2 diabetics had higher apo J concentrations (men, 83.1+/-3.4 microg/mL, n=64; women, 64.0+/-2.3 microg/mL, n=46) than healthy men and women (p<0.001). In these Type 2 diabetics, apo J concentration was unrelated to PON1 concentration, but was positively related to blood glucose (p<0.01). After adjustment for its relation to blood glucose, the mean apo J concentration was similar in diabetics and healthy subjects. These findings suggest that apo J may be anti-atherogenic in humans, and that its concentration is raised by Type 2 diabetes.
Assuntos
Clusterina/sangue , Doença das Coronárias/sangue , Diabetes Mellitus Tipo 2/sangue , Ensaio de Imunoadsorção Enzimática/métodos , Anticorpos Monoclonais/imunologia , Clusterina/imunologia , Ensaio de Imunoadsorção Enzimática/normas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Valores de ReferênciaRESUMO
Apolipoprotein A-V (apoA-V) is a recently discovered apolipoprotein that appears to have a role in plasma triglyceride (TG) transport. We have developed an ELISA for apoA-V using monoclonal antibodies that has a lower limit of detection of 0.3 ng/ml and linearity up to 20 ng/ml. The ELISA was then used to quantify plasma apoA-V in 196 healthy subjects and 106 patients with insulin-resistant diabetes mellitus. In the healthy subjects, total apoA-V concentration was 179.2 +/- 74.8 ng/ml, and it was greater in females than in males (P < 0.005). It was correlated positively with the plasma HDL cholesterol (r = 0.32, P < 0.0001), apoA-I (r = 0.27, P = 0.0001), and apoE (r = 0.18, P = 0.011) concentrations and negatively with plasma TG concentration (r = -0.22, P = 0.021). In relation to single nucleotide polymorphism 3 (-1131C/T) of the apoA-V gene, apoA-V concentration was higher in the T/T type than in the C/C type (P < 0.01). Plasma TG concentration was lower in the T/T type than in the C/C or C/T type (P < 0.05). ApoA-V concentration was lower in the diabetic patients (69.4 +/- 44.3 ng/ml; P < 0.01) than in the healthy controls.
Assuntos
Apolipoproteínas/sangue , Ensaio de Imunoadsorção Enzimática/métodos , Anticorpos Monoclonais , Apolipoproteína A-I/sangue , Apolipoproteína A-V , Apolipoproteínas/genética , Apolipoproteínas A , Apolipoproteínas E/sangue , HDL-Colesterol/sangue , Diabetes Mellitus Tipo 2/sangue , Feminino , Humanos , Masculino , Polimorfismo de Nucleotídeo Único , Valores de Referência , Caracteres Sexuais , Triglicerídeos/sangueRESUMO
Hyperlipidemia is a common feature of diabetes and is related to cardiovascular disease. The very low-density lipoprotein receptor (VLDL-R) is a member of the low-density lipoprotein receptor (LDL-R) family. It binds and internalizes triglyceride-rich lipoproteins with high specificity. We examined the etiology of hyperlipidemia in the insulin-deficient state. VLDL-R expression in heart and skeletal muscle were measured in rats with streptozotocin (STZ)-induced diabetes. STZ rats showed severe hyperlipidemia on d 21 and 28, with a dramatic decline in VLDL-R protein in skeletal muscle (>90%), heart (approximately 50%) and a loss of adipose tissues itself on d 28. The reduction of VLDL-R protein in skeletal muscle could not be explained simply by a decrease at the transcriptional level, because a dissociation between VLDL-R protein and mRNA expression was observed. The expression of LDL-R and LDL-R-related protein in liver showed no consistent changes. Furthermore, no effect on VLDL-triglyceride production in liver was observed in STZ rats. A decrease in postheparin plasma lipoprotein lipase activity started on d 7 and continued to d 28 at the 50% level even though severe hyperlipidemia was detected only on d 21 and 28. In rat myoblast cells, serum deprivation for 24 h induced a reduction in VLDL-R proteins. Insulin (10(-6) m), but not IGF-I (10 ng/ml), restored the decreased VLDL-R proteins by serum deprivation. These results suggest that the combination of VLDL-R deficiency and reduced plasma lipoprotein lipase activity may be responsible for severe hyperlipidemia in insulin-deficient diabetes.
Assuntos
Diabetes Mellitus Experimental/fisiopatologia , Insulina/fisiologia , Receptores de LDL/fisiologia , Tecido Adiposo/fisiopatologia , Animais , HDL-Colesterol/sangue , LDL-Colesterol/sangue , VLDL-Colesterol/sangue , Coração/fisiopatologia , Hiperlipidemias/etiologia , Lipoproteínas/metabolismo , Masculino , Músculo Esquelético/fisiopatologia , RNA/genética , RNA/isolamento & purificação , Ratos , Ratos Sprague-Dawley , Receptores de LDL/deficiência , Receptores de LDL/genética , Valores de Referência , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Triglicerídeos/metabolismoRESUMO
We have previously shown that intravenous apolipoprotein (apo) A-I/phosphatidylcholine (apo A-I/PC) discs increase plasma high-density lipoprotein (HDL) concentration in humans. We have now studied the associated changes in two enzymes, paraoxonase (PON) and platelet-activating factor acetylhydrolase (PAF-AH) that are carried in whole or in part by HDLs, and are thought to influence atherogenesis by hydrolyzing oxidized phospholipids in lipoproteins. Apo A-I/PC discs (40 mg/kg over 4 h) were infused into eight healthy males. Although plasma apo A-I and HDL cholesterol increased on average by 178 and 158%, respectively, plasma total PON and total PAF-AH concentrations did not rise. By the end of the infusion, HDL-associated PAF-AH had increased by 0.56 +/- 0.14 microg/mL (mean +/- S.D., P < 0.01), and nonHDL-associated PAF-AH had decreased by 0.84 +/- 0.11 microg/mL (P < 0.05). These changes were accompanied by an increase in the HDL-associated PAF-AH/apo A-I ratio from 0.19 to 0.35 (P < 0.05), and by a decrease in the nonHDL-associated PAF-AH/apo B ratio from 2.1 to 1.4 (P < 0.05). No changes in PON or PAF-AH concentrations were detected in prenodal lymph (tissue fluid), collected continuously from the leg. Our results show that the total concentrations of PON and PAF-AH in plasma are uninfluenced by plasma HDL concentration. PAF-AH transfers readily between HDLs and LDLs in vivo, and its distribution between them is determined partly by their relative concentrations and partly by HDL composition.
Assuntos
1-Alquil-2-acetilglicerofosfocolina Esterase/sangue , Apolipoproteína A-I/administração & dosagem , Arteriosclerose/tratamento farmacológico , Arildialquilfosfatase/sangue , HDL-Colesterol/sangue , Fosfatidilcolinas/administração & dosagem , Adulto , Apolipoproteína A-I/sangue , Arteriosclerose/prevenção & controle , LDL-Colesterol/sangue , Ativação Enzimática/efeitos dos fármacos , Humanos , Injeções Intravenosas , Linfa/enzimologia , Masculino , Fosfatidilcolinas/sangueRESUMO
Plasma cholesteryl ester transfer protein (CETP) facilitates the transfer of cholesteryl ester (CE) from high density lipoprotein (HDL) to apolipoprotein B-containing lipoproteins. Since CETP regulates the plasma levels of HDL cholesterol and the size of HDL particles, CETP is considered to be a key protein in reverse cholesterol transport (RCT), a protective system against atherosclerosis. The importance of plasma CETP in lipoprotein metabolism was demonstrated by the discovery of CETP-deficient subjects with marked hyperalphalipoproteinemia (HALP). Genetic CETP deficiency is the most important and common cause of HALP in the Japanese. Ten mutations of the CETP gene have been demonstrated as causes of HALP, including two common mutations: an intron 14 splicing defect (Int14 + 1 G --> A) and an exon 15 missense mutation (D442G). The subjects with CETP deficiency show a variety of abnormalities in the concentration, composition, and function of both HDL and low density lipoprotein (LDL). CETP deficiency is considered a physiological state of impaired RCT, which may possibly lead to the development of atherosclerosis despite high HDL cholesterol levels. However, the pathophysiological significance of CETP in terms of atherosclerosis has been controversial. Epidemiological studies in Japanese-Americans living in Hawaii and Japanese in the Omagari area, where HALP subjects with an intron 14 splicing defect of the CETP gene are markedly frequent, have shown a relatively increased incidence of coronary atherosclerosis in CETP deficiency. On the other hand, the TaqIB polymorphism-B2 allele with low CETP mass and increased HDL cholesterol has been related to a decreased risk for coronary heart disease (CHD) in many studies, including the Framingham Offspring Study. The current review focused on the characterization of the Japanese subjects with CETP deficiency, including our recent findings.
Assuntos
Povo Asiático/genética , Glicoproteínas/deficiência , Hiperlipoproteinemias/genética , Doenças Metabólicas/genética , Adolescente , Adulto , Arteriosclerose/complicações , Proteínas de Transporte/genética , Proteínas de Transferência de Ésteres de Colesterol , Feminino , Glicoproteínas/genética , Humanos , Hiperlipoproteinemias/etiologia , Japão , Metabolismo dos Lipídeos , Masculino , Doenças Metabólicas/complicações , Pessoa de Meia-Idade , Mutação/genética , Polimorfismo Genético/genética , Índice de Gravidade de DoençaRESUMO
Plasma lipoprotein-associated phospholipase A2 (Lp-PLA2), also known as platelet-activating factor (PAF) acetylhydrolase (PAF-AH), is a member of the serine-dependent class of A2 phospholipases that hydrolyze sn2-ester bonds of fragmented or oxidized phospholipids at sites where atherosclerotic plaques are forming. Most circulating Lp-PLA2 is bound to low-density lipoprotein (LDL) particles in plasma and the rest to high-density lipoprotein (HDL). Deficiency of Lp-PLA2 is a predisposing factor for cardiovascular diseases in the Japanese population. We describe here two novel mutations of the gene encoding Lp-PLA2, InsA191 and I317N in Japanese subjects. The first patient, with partial Lp-PLA2 deficiency, was heterozygous for the InsA191 mutation; macrophages from this patient secreted only half the normal amount of Lp-PLA2 in vitro. The other patient, who showed complete Lp-PLA2 deficiency, was a compound heterozygote for the novel I317N mutation and a common V279F mutation; macrophages from that patient failed to secrete any Lp-PLA2. Measurement of Lp-PLA2 mass, activity and Western blotting verified impaired production and secretion of the enzyme after transfection of mutant construct into COS-7 cells. These results indicated that both novel mutants, InsA191 and I317N, impair function of the Lp-PLA2 gene.
Assuntos
Mutação , Fosfolipases A/deficiência , Fosfolipases A/genética , 1-Alquil-2-acetilglicerofosfocolina Esterase , Idoso , Alelos , Animais , Sequência de Bases , Western Blotting , Células COS , Meios de Cultura/farmacologia , Ésteres , Feminino , Heterozigoto , Humanos , Japão , Lipoproteínas LDL/química , Macrófagos/metabolismo , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Monócitos/metabolismo , Mutagênese Sítio-Dirigida , Oxigênio/metabolismo , Fosfolipases A/sangue , Fosfolipases A2 , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Serina/químicaRESUMO
BACKGROUND: In individuals heterozygous for ABCA1 transporter mutations, defective reverse cholesterol transport (RCT) causes low HDL-cholesterol and premature coronary heart disease (CHD). However, the extent to which impaired RCT underlies premature CHD in others with low HDL-cholesterol is not known. The primary acceptors of cell cholesterol are a minor subclass of lipid-poor pre-beta-HDLs. These are generated during remodeling of alpha-HDLs, which account for almost all HDL-cholesterol. We studied the strength of the association of CHD with pre-beta-HDL concentrations in Japanese men. METHODS: Blood was collected from 42 men with clinical CHD and 44 healthy controls 40-70 years of age. Pre-beta-HDL was assayed by crossed immunoelectrophoresis. RESULTS: Cases had lower HDL-cholesterol (-23%), total apolipoprotein A-I (-26%), and pre-beta-HDL (-55%; all P <0.001) concentrations; lower pre-beta-HDL:alpha-HDL ratios (-45%; P <0.001); and higher plasma triglycerides (20%; P <0.03) than the controls. On stepwise logistic regression, CHD was associated most strongly with pre-beta-HDL concentrations. On ROC analysis, pre-beta-HDL concentration discriminated between cases and controls better than any other lipoprotein measurement. When plasma was incubated for 16 h at 37 degrees C, mean (SD) pre-beta-HDL increased by 47 (36)% in controls, but was unchanged in cases (group difference, P <0.001). CONCLUSIONS: Our results suggest that inefficient RCT, secondary to a low pre-beta-HDL concentration and production rate in plasma, contributes to premature CHD in Japanese men with low HDL-cholesterol.
Assuntos
Apolipoproteína A-I/sangue , Doença das Coronárias/sangue , Lipoproteínas HDL/sangue , Adulto , Idoso , Doença das Coronárias/diagnóstico , Lipoproteínas de Alta Densidade Pré-beta , Humanos , Japão , Masculino , Pessoa de Meia-IdadeAssuntos
Arteriosclerose/diagnóstico , Glicoproteínas/sangue , Chaperonas Moleculares/sangue , Doenças Autoimunes/diagnóstico , Biomarcadores/sangue , Clusterina , Glicoproteínas/química , Glicoproteínas/fisiologia , Humanos , Técnicas Imunoenzimáticas/métodos , Nefropatias/diagnóstico , Chaperonas Moleculares/química , Chaperonas Moleculares/fisiologia , Neoplasias/diagnóstico , Doenças Neurodegenerativas/diagnóstico , Valores de Referência , Manejo de EspécimesRESUMO
OBJECTIVE: We have previously shown that intravenous apolipoprotein A-I/phosphatidylcholine (apoA-I/PC) discs increase plasma pre-beta HDL concentration and stimulate reverse cholesterol transport (RCT) in humans. We have now investigated the associated changes in the following 3 HDL components that play key roles in RCT: lecithin:cholesterol acyltransferase (LCAT), cholesteryl ester transfer protein (CETP), and phospholipid transfer protein (PLTP). METHODS AND RESULTS: apoA-I/PC discs (40 mg/kg over 4 hours) were infused into 8 healthy men. Samples of blood and prenodal peripheral lymph were collected for 24 to 48 hours. At 12 hours, plasma LCAT concentration had increased by 0.40+/-0.90 mg/L (+7.8%; mean+/-SD; P<0.05), plasma cholesterol esterification rate by 29.0+/-9.0 nmol/mL per h (+69.5%; P<0.01), plasma CETP concentration by 0.5+/-0.2 mg/L (+29.7%; P<0.01), and plasma PLTP activity by 1.45+/-0.67 micromol/mL per h (+23.9%; P<0.01). In contrast, plasma PLTP concentration had decreased by 4.4+/-2.7 mg/L (-44.8%; P<0.01). The changes in PLTP were accompanied by alterations in the relative proportions of large lipoproteins containing inactive PLTP and small particles containing PLTP of high specific activity. No changes were detected in peripheral lymph. CONCLUSIONS: Nascent HDL secretion may induce changes in PLTP, LCAT, and CETP that promote RCT by catalyzing pre-beta HDL production, cholesterol esterification in HDLs, and cholesteryl ester transfer from HDLs to other lipoproteins.
Assuntos
Apolipoproteína A-I/administração & dosagem , Apolipoproteína A-I/farmacologia , Proteínas Sanguíneas/metabolismo , Glicoproteínas , Linfa/química , Fosfatidilcolinas/administração & dosagem , Fosfatidilcolinas/farmacologia , Proteínas de Transferência de Fosfolipídeos , Adulto , Apolipoproteínas/sangue , Proteínas de Transporte/sangue , Proteínas de Transporte/metabolismo , Proteínas de Transferência de Ésteres de Colesterol , Preparações de Ação Retardada/administração & dosagem , Preparações de Ação Retardada/farmacologia , Sistemas de Liberação de Medicamentos , Humanos , Infusões Intravenosas , Lipídeos/sangue , Lipoproteínas/ultraestrutura , Masculino , Proteínas de Membrana/sangue , Proteínas de Membrana/metabolismo , Microscopia Eletrônica , Fosfatidilcolina-Esterol O-Aciltransferase/sangue , Fosfatidilcolina-Esterol O-Aciltransferase/metabolismoRESUMO
Platelet-activating factor acetylhydrolase (PAF-AH) is a phospholipase A2 associated with lipoproteins that hydrolyzes platelet-activating factor (PAF) and oxidized phospholipids. We have developed an ELISA for PAF-AH that is more sensitive than previous methods, and have quantified HDL-associated and non-HDL-associated PAF-AH in healthy, hyperlipidemic, and diabetic subjects. In healthy subjects, plasma total PAF-AH concentration was positively correlated with PAF-AH activity and with plasma total cholesterol, triacylglycerol, LDL cholesterol and apolipoprotein B (apoB) concentrations (all P < 0.01). HDL-associated PAF-AH concentration was correlated positively with plasma apoA-I and HDL cholesterol. Subjects with hyperlipidemia (n = 73) and diabetes mellitus (n = 87) had higher HDL-associated PAF-AH concentrations than did controls (P < 0.01). Non-HDL-associated PAF-AH concentration was lower in diabetic subjects than in controls (P < 0.01). Both hyperlipidemic and diabetic subjects had lower ratios of PAF-AH to apoB (P < 0.01) and higher ratios of PAF-AH to apoA-I (P < 0.01) than did controls. Our results show that the distribution of PAF-AH mass between HDLs and LDLs is determined partly by the concentrations of the lipoproteins and partly by the mass of enzyme per lipoprotein particle, which is disturbed in hyperlipidemia and diabetes mellitus.
Assuntos
1-Alquil-2-acetilglicerofosfocolina Esterase/sangue , HDL-Colesterol/sangue , Diabetes Mellitus/sangue , Ensaio de Imunoadsorção Enzimática/métodos , Hiperlipidemias/sangue , Lipoproteínas/sangue , 1-Alquil-2-acetilglicerofosfocolina Esterase/genética , 1-Alquil-2-acetilglicerofosfocolina Esterase/metabolismo , Anticorpos Monoclonais/imunologia , Apoproteínas/sangue , Sequência de Bases , Colesterol/sangue , LDL-Colesterol/sangue , Diabetes Mellitus/genética , Diabetes Mellitus/metabolismo , Feminino , Técnicas Genéticas , Humanos , Hiperlipidemias/genética , Hiperlipidemias/metabolismo , Lipoproteínas/classificação , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Fosfolipases A/sangue , Fosfolipases A/metabolismo , Fosfolipases A2 , Fator de Ativação de Plaquetas/metabolismo , Triglicerídeos/sangueRESUMO
Plasma phospholipid transfer protein (PLTP) plays an important role in lipoprotein metabolism and reverse cholesterol transport. We have recently reported that plasma PLTP concentration correlates positively with plasma HDL cholesterol (HDL-C) but not with PLTP activity in healthy subjects. We have also shown that PLTP exists as active and inactive forms in healthy human plasma. In the present study, we measured plasma PLTP concentration and PLTP activity, and analyzed the distribution of PLTP in normolipidemic subjects (controls), cholesteryl ester transfer protein (CETP) deficiency, and hypo-alphalipoproteinemia (hypo-ALP). Plasma PLTP concentration was significantly lower (0.7 +/- 0.4 mg/l, mean +/- SD, n = 9, P < 0.001) in the hypo-ALP subjects, and significantly higher (19.5 +/- 4.3 mg/l, n = 17, P < 0.001) in CETP deficiency than in the controls (12.4 +/- 2.3 mg/l, n = 63). In contrast, we observed no significant differences in plasma PLTP activity between controls, hypo-ALP subjects, and CETP deficiency (6.2 +/- 1.3, 6.1 +/- 1.8, and 6.8 +/- 1.2 micro mol/ml/h, respectively). There was a positive correlation between PLTP concentration and plasma HDL-C (r = 0.81, n = 89, P < 0.001). By size exclusion chromatography analysis, we found that the larger PLTP containing particles without PLTP activity (inactive form of PLTP) were almost absent in the plasma of hypo-ALP subjects, and accumulated in the plasma of CETP deficiency compared with those of controls. These results indicate that the differences in plasma PLTP concentrations between hypo-ALP subjects, CETP deficiency, and controls are mainly due to the differences in the amount of the inactive form of PLTP.
Assuntos
Proteínas de Transporte/sangue , HDL-Colesterol/sangue , Hiperlipoproteinemias/sangue , Proteínas de Membrana/sangue , Proteínas de Transferência de Fosfolipídeos , Adulto , Idoso , Western Blotting , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Cholesteryl ester transfer protein (CETP) deficiency is one of the most important and common causes of hyperalphalipoproteinemia (HALP) in the Japanese. CETP deficiency is thought to be a state of impaired reverse cholesterol transport, which may possibly lead to the development of atherosclerotic cardiovascular disease despite high HDL-cholesterol (HDL-C) levels. Thus, it is important to investigate whether HALP is caused by CETP deficiency. In the present study, we identified two novel missense mutations in the CETP gene among 196 subjects with a marked HALP (HDL-C > or = 2.59 mmol/l = 100 mg/dl). The two missense mutations, L151P (CTC-->CCC in exon 5) and R282C (CGC-->TGC in exon 9), were found in compound heterozygous subjects with D442G mutation, whose plasma CETP levels were significantly lower when compared with those in D442G heterozygous subjects. In COS-7 cells expressing the wild type and mutant CETP, these two mutant CETP showed a marked reduction in the secretion of CETP protein into media (0% and 39% of wild type for L151P and R282C, respectively). These results suggested that two novel missense mutations cause the decreased secretion of CETP protein into circulation leading to HALP. By using the Invader assay for seven mutations, including two novel mutations of the CETP gene, we investigated their frequency among 466 unrelated subjects with HALP (HDL-C > or = 2.07 mmol/l = 80 mg/dl). Two novel mutations were rare, but L151P mutation was found in unrelated subjects with a marked HALP. Furthermore, we demonstrated that CETP deficiency contributes to 61.7% and 31.4% of marked HALP and moderate HALP in the Japanese, respectively.
Assuntos
Proteínas de Transporte/genética , Glicoproteínas , Hiperlipoproteinemias/genética , Mutação de Sentido Incorreto/genética , Adulto , Idoso , Sequência de Aminoácidos , Sequência de Bases , Proteínas de Transporte/química , Proteínas de Transferência de Ésteres de Colesterol , HDL-Colesterol/sangue , Feminino , Genótipo , Humanos , Hiperlipoproteinemias/sangue , Hiperlipoproteinemias/etnologia , Japão/etnologia , Masculino , Pessoa de Meia-Idade , Dados de Sequência MolecularRESUMO
Despite progress in molecular characterization, specific diagnoses of disorders belonging to a group of inherited hypoalphalipoproteinemias, i.e., apolipoprotein AI deficiency, lecithin-cholesterol acyltransferase deficiency, Tangier disease (TD), and familial high-density lipoprotein (HDL) deficiency, remain difficult on a purely clinical basis. Several TD patients were recently found to be homozygous for mutations in the ABCA1 gene. We have documented here a clinical variant of TD in a Japanese patient who manifested corneal lipidosis and premature coronary artery disease as well as an almost complete absence of HDL-cholesterol, by identifying a novel homozygous ABCA1 mutation (R1680W). We propose that patients with apparently isolated HDL deficiency who are found to carry ABCA1 mutations may in fact belong to a category of TD patients whose phenotypic features are only partially expressed, and that a number of hidden clinical variants of TD might exist among other HDL deficiency patients who have escaped correct clinical diagnosis.