RESUMO
AIMS: To determine whether alanine aminotransferase or gamma-glutamyltransferase levels, as markers of liver health and non-alcoholic fatty liver disease, might predict cardiovascular events in people with Type 2 diabetes. METHODS: Data from the Fenofibrate Intervention and Event Lowering in Diabetes study were analysed to examine the relationship between liver enzymes and incident cardiovascular events (non-fatal myocardial infarction, stroke, coronary and other cardiovascular death, coronary or carotid revascularization) over 5 years. RESULTS: Alanine aminotransferase measure had a linear inverse relationship with the first cardiovascular event occurring in participants during the study period. After adjustment, for every 1 sd higher baseline alanine aminotransferase measure (13.2 U/l), the risk of a cardiovascular event was 7% lower (95% CI 4-13; P = 0.02). Participants with alanine aminotransferase levels below and above the reference range 8-41 U/l for women and 9-59 U/l for men, had hazard ratios for a cardiovascular event of 1.86 (95% CI 1.12-3.09) and 0.65 (95% CI 0.49-0.87), respectively (P = 0.001). No relationship was found for gamma-glutamyltransferase. CONCLUSIONS: The data may indicate that in people with Type 2 diabetes, which is associated with higher alanine aminotransferase levels because of prevalent non-alcoholic fatty liver disease, a low alanine aminotransferase level is a marker of hepatic or systemic frailty rather than health.
Assuntos
Alanina Transaminase/sangue , Doenças Cardiovasculares/epidemiologia , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/epidemiologia , Fenofibrato/uso terapêutico , Adulto , Idoso , Biomarcadores/sangue , Doenças Cardiovasculares/sangue , Doenças Cardiovasculares/etiologia , Diabetes Mellitus Tipo 2/complicações , Feminino , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Fatores de Risco , gama-Glutamiltransferase/sangueRESUMO
More than half of the patients with angiographically confirmed premature coronary heart disease (CHD) have a familial lipoprotein disorder. Familial combined hyperlipidaemia (FCHL) represents the most common genetic dyslipidemia with a prevalence of 1.0-2.0%. FCHL is estimated to cause 10-20% of premature CHD and is characterized by elevated levels of cholesterol, triglycerides, or both. Attempts to characterize genes predisposing to FCHL have been hampered by its equivocal phenotype definition, unknown mode of inheritance and genetic heterogeneity. In order to minimize genetic heterogeneity, we chose 31 extended FCHL families from the isolated Finnish population that fulfilled strictly defined criteria for the phenotype status. We performed linkage analyses with markers from ten chromosomal regions that contain lipid-metabolism candidate genes. One marker, D1S104, adjacent to the apolipoprotein A-II (APOA2) gene on chromosome 1, revealed a lod score of Z = 3.50 assuming a dominant mode of inheritance. Multipoint analysis combining information from D1S104 and the neighbouring marker D1S1677 resulted in a lod score of 5.93. Physical positioning of known genes in the area (APOA2 and three selectin genes) outside the linked region suggests a novel locus for FCHL on 1q21-q23. A second paper in this issue (Castellani et al.) reports the identification of a mouse combined hyperlipidaemia locus in the syntenic region of the mouse genome, thus further implicating a gene in this region in the aetiology of FCHL.
Assuntos
Cromossomos Humanos Par 1/genética , Hiperlipidemias/genética , Adulto , Idoso , Mapeamento Cromossômico , Saúde da Família , Feminino , Genes/genética , Ligação Genética , Marcadores Genéticos/genética , Humanos , Escore Lod , Masculino , Pessoa de Meia-IdadeRESUMO
AIMS/HYPOTHESIS: We investigated effects of renal function and albuminuria on cardiovascular outcomes in 9,795 low-risk patients with diabetes in the Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) study. METHODS: Baseline and year 2 renal status were examined in relation to clinical and biochemical characteristics. Outcomes included total cardiovascular disease (CVD), cardiac and non-cardiac death over 5 years. RESULTS: Lower estimated GFR (eGFR) vs eGFR ≥90 ml min⻹ 1.73 m⻲ was a risk factor for total CVD events: (HR [95% CI] 1.14 [1.01-1.29] for eGFR 60-89 ml min⻹ 1.73 m⻲; 1.59 [1.28-1.98] for eGFR 30-59 ml min⻹ 1.73 m⻲; p < 0.001; adjusted for other characteristics). Albuminuria increased CVD risk, with microalbuminuria and macroalbuminuria increasing total CVD (HR 1.25 [1.01-1.54] and 1.19 [0.76-1.85], respectively; p = 0.001 for trend) when eGFR ≥90 ml min⻹ 1.73 m⻲. CVD risk was further modified by renal status changes over the first 2 years. In multivariable analysis, 77% of the effect of eGFR and 81% of the effect of albumin:creatinine ratio were accounted for by other variables, principally low HDL-cholesterol and elevated blood pressure. CONCLUSIONS/INTERPRETATION: Reduced eGFR and albuminuria are independent risk factors for cardiovascular events and mortality rates in a low-risk population of mainly European ancestry. While their independent contributions to CVD risk appear small when other risk factors are considered, they remain excellent surrogate markers in clinical practice because they capture risk related to a number of other characteristics. Therefore, both should be considered when assessing prognosis and treatment strategies in patients with diabetes, and both should be included in risk models.
Assuntos
Albuminúria/fisiopatologia , Doenças Cardiovasculares/etiologia , Diabetes Mellitus Tipo 2/mortalidade , Diabetes Mellitus Tipo 2/fisiopatologia , Fenofibrato/uso terapêutico , Taxa de Filtração Glomerular/fisiologia , Hipolipemiantes/uso terapêutico , Idoso , Doenças Cardiovasculares/fisiopatologia , Diabetes Mellitus Tipo 2/tratamento farmacológico , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
AIMS/HYPOTHESIS: The apolipoprotein B (ApoB):apolipoprotein A (ApoA)-I ratio may be a better indicator of cardiovascular disease (CVD) risk in people with type 2 diabetes than traditional lipid risk markers (LDL-cholesterol, HDL-cholesterol and triacylglycerol), but whether the ApoB:ApoA-I ratio should be used to indicate lipid-lowering therapy is still debated. METHODS: The Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) study randomised 9,795 patients with type 2 diabetes to fenofibrate (200 mg daily) or placebo and followed them up for a median of 5 years. We compared ApoB, ApoA-I, ApoAII and the ApoB:ApoA-I ratio with traditional lipid variables as predictors of CVD risk. We estimated the HR of the effect of 1 SD difference in baseline concentrations of lipids, apolipoproteins and respective ratios on the risk of CVD events and also used receiver operating characteristic curve analysis. RESULTS: In the placebo group, the variables best predicting CVD events were non-HDL-cholesterol:HDL-cholesterol, total cholesterol:HDL-cholesterol (HR 1.21, p < 0.001 for both), ApoB:ApoA-I (HR 1.20, p < 0.001), LDL-cholesterol:HDL-cholesterol (HR 1.17, p < 0.001), HDL-cholesterol (HR 0.84, p < 0.001) and ApoA-I (HR 0.85, p < 0.001). In the fenofibrate group, the first four predictors were very similar (but ApoB:ApoA-I was fourth), followed by non-HDL-cholesterol and ApoB. Lipid ratios and ApoB:ApoA-I performed better than any single lipid or apolipoprotein in predicting CVD risk. CONCLUSIONS/INTERPRETATION: In patients with type 2 diabetes in the FIELD study, traditional lipid ratios were as strong as the ApoB:ApoA-I ratio in predicting CVD risk. The data provide little evidence for replacement of traditional lipids and their ratios with measures of ApoB, ApoA-I and their ratio.
Assuntos
Apolipoproteínas/metabolismo , Diabetes Mellitus Tipo 2/sangue , Lipídeos/sangue , Idoso , Apolipoproteína A-I/metabolismo , Apolipoproteína A-II/sangue , Apolipoproteínas B/metabolismo , Doenças Cardiovasculares/sangue , Doenças Cardiovasculares/tratamento farmacológico , Doenças Cardiovasculares/metabolismo , Colesterol/sangue , HDL-Colesterol/sangue , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Feminino , Fenofibrato/uso terapêutico , Humanos , Hipolipemiantes/uso terapêutico , Masculino , Pessoa de Meia-Idade , Fatores de Risco , Triglicerídeos/sangueRESUMO
Relationship between the efficiency of cholesterol absorption and apolipoprotein E (apoE) phenotype was studied in a random sample of middle-aged Finnish men. Subjects that were either heterozygous or homozygous for the allele epsilon 2 absorbed less and synthesized more cholesterol than those with the phenotype E4/3 and E4/4, the values for the individuals with the most frequent phenotype E3/3 (56% of the population sample) falling in between. Among the whole study group, the sum of the subscripts of apoE phenotype (e.g., E2/3 = 5) was correlated positively with the fractional absorption of cholesterol (r = 0.40; P less than 0.05) and negatively with the serum level of lathosterol, a cholesterol precursor sterol reflecting the activity of cholesterol synthesis (r = -0.48; P less than 0.01). Thus, apoE polymorphism appears to affect the efficiency of cholesterol absorption and may by this mechanism contribute to the variation in plasma total and low density lipoprotein cholesterol concentration.
Assuntos
Apolipoproteínas E/fisiologia , Colesterol/metabolismo , Absorção Intestinal , Adulto , Ácidos e Sais Biliares/metabolismo , Colesterol/sangue , Doença das Coronárias/metabolismo , Humanos , Pessoa de Meia-Idade , Fenótipo , Receptores de LDL/fisiologiaRESUMO
The human paraoxonase gene (HUMPONA) is codominantly expressed as alleles A and G. The A allele codes for glutamine (A genotype) and the G allele for arginine (B genotype) at position 191 of the paraoxonase enzyme. This genetic polymorphism has been suggested to be associated with the predisposition to coronary artery disease (CAD). We investigated the frequency of paraoxonase A and G alleles in 380 well-characterized CAD patients and in 169 controls. The most common genotype in both the patients with CAD (211/380) and in healthy Finnish individuals (87/169) was AA (Gln/Gln). The heterozygous AM (Gln/Arg) genotype was present in 140 of the patients and in 75 controls. The frequency of the A allele was 0.74 in both patients and controls. The genotype distribution between the two groups did not differ (P = 0.12, chi2 test). The genotype distributions were also similar to those reported earlier in other caucasoid populations. In conclusion, we found no association between the Gln-Arg 191 polymorphism of the human paraoxonase gene and coronary artery disease in Finns.
Assuntos
Doença das Coronárias/genética , Esterases/genética , Adulto , Alelos , Arildialquilfosfatase , Sequência de Bases , Primers do DNA/química , Feminino , Finlândia/etnologia , Frequência do Gene , Humanos , Masculino , Dados de Sequência Molecular , Polimorfismo GenéticoRESUMO
A new apolipoprotein E (apo E) phenotype has been demonstrated in a Finnish hypertriglyceridemic subject (R.M.). At the time of this study, R.M.'s plasma triglyceride and cholesterol levels were 1,021 and 230 mg/dl, respectively. The subject's apo E isoelectric focusing pattern was characterized by two major bands, one in the E3 position and the other in the E1 position. Normally the E1 position is occupied by sialylated derivatives of apo E4, E3, or E2. The E1 band of subject R.M. is not a sialylated form, however, because it was not affected by neuraminidase digestion. The identity of the E1 variant as a genetically determined structure was established by amino acid and partial sequence analyses, confirming that the variant is an example of a previously uncharacterized apo E phenotype, E3/1. Both cysteamine modification and amino acid analysis demonstrated that this variant contains two cysteine residues per mole. Sequence analysis of two cyanogen bromide fragments and one tryptic fragment of the apo E3/1 showed that it differs from E2(Arg158----Cys) at residue 127, where an aspartic acid residue is substituted for glycine. This single amino acid interchange is sufficient to account for the one-charge difference observed on isoelectric focusing gels between E2(Arg158----Cys) and the E1 variant. The variant has been designated E1 (Gly127----Asp, Arg158----Cys). When compared with apo E3, the E1 variant demonstrated reduced ability to compete with 125I-LDL for binding to LDL (apo B,E) receptors on cultured fibroblasts (approximately 4% of the amount of binding of apo E3). This defective binding is similar to that of E2-(Arg158----Cys). Therefore, the binding defect of the variant is probably due to the presence of cysteine at residue 158, rather than aspartic acid at residue 127. In contrast, the apo E3 isoform from this subject demonstrated normal binding activity, indicating that it has a normal structure. In family studies, the vertical transmission of the apo E1 variant has been established. It is not yet clear, however, if the hypertriglyceridemia observed in the proband is associated with the presence of the E1(Gly127----Asp, Arg158----Cys) variant.
Assuntos
Apolipoproteínas E , Apolipoproteínas/sangue , Variação Genética , Hiperlipoproteinemia Tipo IV/sangue , Adolescente , Sequência de Aminoácidos , Aminoácidos/sangue , Apolipoproteínas/genética , Apolipoproteínas/isolamento & purificação , Criança , Cisteamina , Feminino , Humanos , Hiperlipoproteinemia Tipo IV/genética , Focalização Isoelétrica , Masculino , Pessoa de Meia-Idade , Fenótipo , Receptores de Superfície Celular/análise , Receptores de LDLRESUMO
Salla disease is a lysosomal storage disorder characterized by mental retardation and disturbed sialic acid metabolism. To study endogenous synthesis and breakdown of sialic acid, fibroblasts were incubated for 5 d in the presence and then in the absence of N-[3H]acetylmannosamine. Labeling of free sialic acid was 5-10 times higher in mutant than in normal cells. Radioactivity decreased in 4 d by 75% in normal but only by 30% in mutant fibroblasts. The labeling pattern was not normalized upon coculture of mutant and normal cells. To study the metabolism of extracellular sialic acid, low-density lipoprotein (LDL) was labeled in the sialic acid moiety (periodate-NaB3H4) or in the protein moiety (125I). Binding, internalization, lysosomal degradation, and exit of products of protein catabolism were similar in normal and mutant fibroblasts. Upon incubation with LDL labeled in the sialic acid moiety, mutant cells accumulated 2-3 times more free sialic acid radioactivity than normal fibroblasts, mostly in the lysosomal fraction. After a 24-h chase incubation, radioactivity in free sialic acid decreased by 70-80% in normal but only by 10-30% in mutant cells. In mutant fibroblasts, 40% of the radioactivity remained in lysosomes, whereas no labeled free sialic acid was detected in lysosomes from normal fibroblasts. We conclude that in Salla disease, fibroblast endogenous synthesis of sialic acid and lysosomal cleavage of exogenous glycoconjugates is normal, but free sialic acid cannot leave the lysosome. These findings suggest that the basic defect in Salla disease is deficient transport of free sialic acid through the lysosomal membrane.
Assuntos
Fibroblastos/metabolismo , Lisossomos/metabolismo , Erros Inatos do Metabolismo/metabolismo , Ácidos Siálicos/metabolismo , Células Cultivadas , Hexosaminas/metabolismo , Humanos , Lipoproteínas LDL/metabolismo , Ácido N-Acetilneuramínico , TrítioRESUMO
The associations between six genetic polymorphisms in the hepatic lipase (HL) gene (LIPC) and variation in postheparin HL activity and fasting serum lipoproteins were evaluated in 395 male Finnish coronary heart disease patients with HDL cholesterol concentrations = 1.1 mmol/liter. The LIPC promoter polymorphism at position -514 was highly significantly associated with variation in HL activity (P = 0.0000008), with mean activities of 20.4, 17.5, and 13.2 mumol free fatty acid/ml per hour in subjects having C/C, C/T, and T/T genotypes, respectively. Furthermore, the triglyceride content of low density lipoprotein, intermediate density lipoprotein and HDL, and the cholesterol content of intermediate density lipoprotein were found to be associated with variation at LIPC position -514. However, there was no association of this polymorphism with coronary heart disease. These data suggest that the LIPC promoter variation is likely to be the basis for variation in HL activity, which underlies the variation in serum lipoprotein phenotypes in this sample.
Assuntos
Doença das Coronárias/genética , Doença das Coronárias/prevenção & controle , Lipase/genética , Fígado/enzimologia , Polimorfismo Genético , Regiões Promotoras Genéticas , Colesterol/análise , Colesterol/metabolismo , Doença das Coronárias/enzimologia , DNA/análise , DNA/genética , Ácidos Graxos/análise , Ácidos Graxos/metabolismo , Haplótipos , Humanos , Lipase/metabolismo , Lipase Lipoproteica/metabolismo , Lipoproteínas HDL/análise , Lipoproteínas HDL/metabolismo , Lipoproteínas LDL/análise , Lipoproteínas LDL/metabolismo , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Triglicerídeos/análise , Triglicerídeos/metabolismoRESUMO
BACKGROUND: Patients with type 2 diabetes mellitus are at increased risk of cardiovascular disease, partly owing to dyslipidaemia, which can be amenable to fibrate therapy. We designed the Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) study to assess the effect of fenofibrate on cardiovascular disease events in these patients. METHODS: We did a multinational, randomised controlled trial with 9795 participants aged 50-75 years, with type 2 diabetes mellitus, and not taking statin therapy at study entry. After a placebo and a fenofibrate run-in phase, we randomly assigned patients (2131 with previous cardiovascular disease and 7664 without) with a total-cholesterol concentration of 3.0-6.5 mmol/L and a total-cholesterol/HDL-cholesterol ratio of 4.0 or more or plasma triglyceride of 1.0-5.0 mmol/L to micronised fenofibrate 200 mg daily (n=4895) or matching placebo (n=4900). Our primary outcome was coronary events (coronary heart disease death or non-fatal myocardial infarction); the outcome for prespecified subgroup analyses was total cardiovascular events (the composite of cardiovascular death, myocardial infarction, stroke, and coronary and carotid revascularisation). Analysis was by intention to treat. The study was prospectively registered (number ISRCTN 64783481). FINDINGS: Vital status was confirmed on all but 22 patients. Averaged over the 5 years' study duration, similar proportions in each group discontinued study medication (10% placebo vs 11% fenofibrate) and more patients allocated placebo (17%) than fenofibrate (8%; p<0.0001) commenced other lipid treatments, predominantly statins. 5.9% (n=288) of patients on placebo and 5.2% (n=256) of those on fenofibrate had a coronary event (relative reduction of 11%; hazard ratio [HR] 0.89, 95% CI 0.75-1.05; p=0.16). This finding corresponds to a significant 24% reduction in non-fatal myocardial infarction (0.76, 0.62-0.94; p=0.010) and a non-significant increase in coronary heart disease mortality (1.19, 0.90-1.57; p=0.22). Total cardiovascular disease events were significantly reduced from 13.9% to 12.5% (0.89, 0.80-0.99; p=0.035). This finding included a 21% reduction in coronary revascularisation (0.79, 0.68-0.93; p=0.003). Total mortality was 6.6% in the placebo group and 7.3% in the fenofibrate group (p=0.18). Fenofibrate was associated with less albuminuria progression (p=0.002), and less retinopathy needing laser treatment (5.2%vs 3.6%, p=0.0003). There was a slight increase in pancreatitis (0.5%vs 0.8%, p=0.031) and pulmonary embolism (0.7%vs 1.1%, p=0.022), but no other significant adverse effects. INTERPRETATION: Fenofibrate did not significantly reduce the risk of the primary outcome of coronary events. It did reduce total cardiovascular events, mainly due to fewer non-fatal myocardial infarctions and revascularisations. The higher rate of starting statin therapy in patients allocated placebo might have masked a moderately larger treatment benefit.
Assuntos
Doenças Cardiovasculares/prevenção & controle , Colesterol/sangue , Diabetes Mellitus Tipo 2/complicações , Dislipidemias/tratamento farmacológico , Fenofibrato/uso terapêutico , Hipolipemiantes/uso terapêutico , Triglicerídeos/sangue , Idoso , Doenças Cardiovasculares/etiologia , Doença das Coronárias/mortalidade , Doença das Coronárias/prevenção & controle , Diabetes Mellitus Tipo 2/sangue , Método Duplo-Cego , Dislipidemias/complicações , Feminino , Fenofibrato/efeitos adversos , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Hipolipemiantes/efeitos adversos , Masculino , Pessoa de Meia-IdadeRESUMO
The interaction of recombinant apo(a) (r-apo(a)) with low-density lipoprotein (LDL) has been examined using ultracentrifugation and affinity chromatography. R-apo(a) forms a non-covalent complex with human LDL. This LDL-r-apo(a) complex, reconstituted Lp(a), r-Lp(a), which can be isolated by ultracentrifugation, has protease activity. The protease activity reached maximum at an equimolar ratio of r-apo(a) and LDL. Proline and epsilon aminocaproic acid (at a concentration of 50 mM) caused dissociation of r-Lp(a) and simultaneous loss of enzyme activity. Mouse LDL that did not form a complex with r-apo(a) did not activate the protease region of r-apo(a). Unlike plasma Lp(a), r-Lp(a) was dissociated during affinity chromatography on Lysine-Sepharose. This dissociation led to loss of enzyme activity. We conclude that the formation of a non-covalent complex between r-apo(a) and LDL leads to activation of the protease region of r-apo(a). The results suggest that non-covalent binding between r-apo(a) and LDL is a pre-requisite for the enzyme activity of the protease region of r-apo(a).
Assuntos
Apolipoproteínas A/metabolismo , Lipoproteínas LDL/farmacologia , Serina Endopeptidases/metabolismo , Animais , Apolipoproteínas A/química , Ativação Enzimática , Humanos , Camundongos , Proteínas Recombinantes/metabolismo , Serina Endopeptidases/química , Inibidores de Serina Proteinase/farmacologiaRESUMO
Lipoprotein lipase has been purified from bovine milk by affinity chromatography on Sepharose containing covalently linked heparin. In addition to an enzyme eluted by salt, further activity could be eluted with detergent. Rechromatography experiments suggested that the two activities were due to the same enzyme. This assumption was further verified by several other criteria as follows: (a) both require a serum activator, (b) their apparent molecular weights (55 000), their amino acid compositions and amino sugar contents were similar and (c) they had identical immunological reactivities. Thus, the enzyme appears to be bound to the heparin-Sepharose matrix by both salt-reversed and detergent-reversed interactions. Sodium deoxycholate stimulated the activity eluted by high salt, but had no effect on the detergent-eluted enzyme.
Assuntos
Lipase Lipoproteica , Leite/enzimologia , Aminoácidos/análise , Animais , Bovinos , Cromatografia de Afinidade , Ácido Desoxicólico/farmacologia , Feminino , Galactosamina/análise , Glucosamina/análise , Cinética , Lipase Lipoproteica/isolamento & purificação , Lipase Lipoproteica/metabolismoRESUMO
1. The uptake of small and large chylomicrons in rat hepatocyte monolayer cultures was compared to the uptake of chylomicron remnants prepared either in vitro with pure milk lipoprotein lipase or in hepatectomized rats. 2. Small chylomicrons (Sf less than 400) markedly inhibited remnant uptake and were taken up more efficiently than large ones (Sf greater than 400), indicating that size may be an important factor for the rate of uptake. The Lineweaver-Burk analysis of the data indicated that the V values for the uptake of both small chylomicrons (Sf less than 400) and of remnants prepared either in hepatectomized rats or in vitro was significantly higher than for chylomicrons with Sf greater than 400, whereas the Km values for the different particles did not differ significantly. 3. Preincubation of chylomicrons with serum caused marked changes in their apolipoprotein composition. A loss of apolipoprotein A-I and an increase in apolipoprotein E content was observed by scanning of SDS-polyacrylamide gels. Th preincubation decreased, however, the subsequent uptake of the chylomicrons. In contrast, the uptake of remnants prepared in vivo, or in vitro with serum present, exceeded that of remnants prepared in vitro with albumin or fetal calf serum as the fatty acid acceptor. 4. The data thus indicate that both the decrease in size and the changes in the particle surface during lipolysis with serum present are likely to contribute to the differences seen in the rate of uptake between native chylomicrons and remnants in hepatocyte monolayers.
Assuntos
Quilomícrons/metabolismo , Fígado/metabolismo , Animais , Apolipoproteínas/metabolismo , Sangue , Células Cultivadas , Quilomícrons/isolamento & purificação , Hepatectomia , Cinética , Lipase Lipoproteica , Masculino , RatosRESUMO
Immunochemical methods for the selective measurement of pig post-heparin plasma lipoprotein lipase and hepatic lipase are described and validated. A simple two step purification method for porcine hepatic lipase from hepatic perfusate based on affinity chromatography and gel filtration is reported. The activity of the post-heparin plasma lipoprotein lipase and hepatic lipase in swine is reported. It is demonstrated that fasting decreases the activity of post-heparin plasma lipoprotein lipase activity more than two-fold while it does not affect the hepatic lipase activity significantly.
Assuntos
Lipase/sangue , Animais , Cromatografia de Afinidade , Jejum , Heparina/farmacologia , Lipase/metabolismo , Lipase Lipoproteica/metabolismo , Fígado/enzimologia , Suínos , TriglicerídeosRESUMO
A natural deficiency of hepatic lipase in rabbits has been exploited to gain insights into the physiological role of this enzyme in the metabolism of plasma lipoproteins. A comparison of human and rabbit lipoproteins revealed obvious species differences in both low-density lipoproteins (LDL) and high-density lipoproteins (HDL), with the rabbit lipoproteins being relatively enlarged, enriched in triacylglycerol and depleted of cholesteryl ester. To test whether these differences related to the low level of hepatic lipase in rabbits, whole plasma or the total lipoprotein fraction from rabbits was either kept at 4 degrees C or incubated at 37 degrees C for 7 h in (i) the absence of lipase, (ii) the presence of hepatic lipase and (iii) the presence of lipoprotein lipase. Following incubation, the lipoproteins were recovered and subjected to gel permeation chromatography to determine the distribution of lipoprotein components across the entire lipoprotein spectrum. An aliquot of the lipoproteins was subjected also to gradient gel electrophoresis to determine the particle size distribution of the LDL and HDL. Both hepatic lipase and lipoprotein lipase hydrolysed lipoprotein triacylglycerol and to a much lesser extent, also phospholipid. There were, however, obvious differences between the enzymes in terms of substrate specificity. In incubations containing hepatic lipase, there was a preferential hydrolysis of HDL triacylglycerol and a lesser hydrolysis of VLDL triacylglycerol. By contrast, lipoprotein lipase acted primarily on VLDL triacylglycerol. When more enzyme was added, both lipases also acted on LDL triacylglycerol, but in no experiment did lipoprotein lipase hydrolyse the triacylglycerol in HDL. Coincident with the hepatic lipase-induced hydrolysis of LDL and HDL triacylglycerol, there were marked reductions in the particle size of both lipoprotein fractions, which were now comparable to those of human LDL and HDL3, respectively.
Assuntos
Lipase/metabolismo , Lipoproteínas HDL/sangue , Lipoproteínas LDL/sangue , Fígado/enzimologia , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Feminino , Heparina/farmacologia , Humanos , Hidrólise , Lipase/deficiência , Lipólise , Lipase Lipoproteica/metabolismo , Lipoproteínas VLDL/sangue , Masculino , Tamanho da Partícula , Especificidade da Espécie , Especificidade por Substrato , Triglicerídeos/metabolismoRESUMO
Phospholipid transfer protein (PLTP) is a plasma protein with two reported in vitro activities: transfer of phospholipids and modulation of HDL particle size. The mechanism of PLTP-mediated phospholipid transfer was studied by determining the acyl chain and headgroup specificity and comparing the results with those obtained with the non-specific lipid transfer protein (ns-LTP), a previously characterised intracellular transfer protein. To verify the results obtained with purified plasma PLTP, recombinant PLTP produced in COS-1 cells was used. The transfer rates were determined by monitoring the transfer of fluorescent, pyrene-labeled phospholipids from quenched donor phospholipid vesicles to HDL3 particles. When the length of the pyrene-labeled acyl chain was varied from 6 to 14 carbons, a fairly monotonous decrease in the transfer rate was observed. No difference in rate was observed for the isomers having the pyrene-labeled and unlabeled acyl chains in reversed positions. PLTP mediated equally the transfer of the various headgroup derivatives except phosphatidylethanolamine (PE), which was transferred 2-3-fold more slowly. In all experiments the plasma and recombinant PLTP behaved identically. The specificity patterns observed for PLTP and ns-LTP were very similar. No PLTP-phospholipid intermediate could be observed, indicating that PLTP, like ns-LTP, does not form a tight complex with the lipid substrate and may thus mediate the transfer of phospholipid via another, yet unspecified mechanism.
Assuntos
Proteínas de Transporte/sangue , Lipoproteínas HDL/sangue , Proteínas de Membrana/sangue , Proteínas de Transferência de Fosfolipídeos , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Animais , Transporte Biológico , Células COS , Células Cultivadas , Clonagem Molecular , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Humanos , Cinética , Lipossomos/metabolismo , Tamanho da Partícula , Pirenos/metabolismo , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Transfecção , TrinitrobenzenosRESUMO
A complement-associated protein SP-40,40, which is a normal constituent of human blood, binds to the main apoprotein, apoA-I, of high density lipoprotein (HDL). This protein, which is identical to apolipoprotein J, was compared to another apoA-I binding protein purified from human placenta. Immunologically the two apoA-I binding proteins are different.
Assuntos
Apolipoproteína A-I/metabolismo , Apolipoproteínas/química , Proteínas de Transporte/química , Glicoproteínas , Lipoproteínas HDL/química , Chaperonas Moleculares , Placenta/química , Sequência de Aminoácidos , Anticorpos Monoclonais , Apolipoproteínas/imunologia , Apolipoproteínas/metabolismo , Proteínas de Transporte/imunologia , Proteínas de Transporte/metabolismo , Clusterina , Feminino , Humanos , Lipoproteínas HDL/imunologia , Lipoproteínas HDL/metabolismo , Dados de Sequência Molecular , Gravidez , Homologia de Sequência do Ácido NucleicoRESUMO
The oxidation of HDL3 by Cu(II) and its effect on the ability of these particles to act as phospholipid acceptors in human plasma phospholipid transfer protein (PLTP)-mediated lipid transfer were investigated. Oxidation of HDL3 was monitored by measuring the following parameters: (i) formation of conjugated dienes, (ii) production of thiobarbituric acid reactive substances (TBARS), (iii) decrease in reactive lysine and (iv) tryptophan residues, (v) change in particle charge and (vi) diameter, and (vii) oligomerisation of apoA-I and apoA-II. Formation of conjugated dienes was the parameter responding to the oxidative treatment with the fastest kinetics. The appearance of TBARS and modification of apolipoprotein tryptophan residues were detected simultaneously but required higher Cu(II) concentrations for maximal kinetics. Cross-linking of the major protein constituents of HDL3, apoA-I and apoA-II, represented later steps of the oxidation process. Further, the oxidative modification was accompanied by a progressive change in HDL3 particle charge and a minor increase in particle diameter. PLTP-mediated phospholipid transfer to the oxidized particles was investigated using an assay measuring the transfer of fluorescent, pyrene-labeled PC. The transfer was significantly inhibited, but only after extensive modification of the HDL proteins, suggesting that the HDL oxidative modifications occurring in vivo do not essentially impair its phospholipid acceptor function. A similar but less pronounced inhibition was observed when two other phospholipid transfer proteins, the nonspecific lipid transfer protein (ns-LTP) and the phosphatidylcholine transfer protein (PC-TP), were studied in parallel. This indicates that the inhibition was partly due to unspecific effects of the modification on acceptor particle surface properties, but included an aspect specific for PLTP.
Assuntos
Proteínas de Transporte/metabolismo , Lipoproteínas HDL/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Transferência de Fosfolipídeos , Fosfolipídeos/metabolismo , Animais , Apolipoproteína A-I/metabolismo , Apolipoproteína A-II/metabolismo , Proteínas de Transporte/sangue , Cobre/metabolismo , Humanos , Técnicas In Vitro , Cinética , Lipoproteínas HDL3 , Proteínas de Membrana/sangue , Oxirredução , Proteínas Recombinantes/metabolismo , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismoRESUMO
High density lipoproteins (HDL) subclasses can be differentiated by two-dimensional non-denaturing polyacrylamide gradient gel electrophoresis (2D-PAGGE) and subsequent immunoblotting. The quantitatively minor HDL-subclasses pre beta 1-LpA-I and gamma-LpE are initial acceptors of cell-derived cholesterol into the plasma compartment. In this study we analysed the effect of phospholipid transfer protein (PLTP) on the electrophoretic distribution of HDL-subclasses in plasma as well as the ability of plasma, pre beta 1-LpA-I, and gamma-LpE to take up [3H]cholesterol from labeled fibroblasts. Pre beta 1-LpA-I but not gamma-LpE disappeared during a 16 hours incubation in the absence of PLTP. During a one minute incubation pre beta 1-LpA-I of pre-incubated plasma released 75% less [3H]cholesterol from radiolabeled fibroblasts than pre beta 1-LpA-I of control plasma. Pre-incubation of plasma reduced the uptake of [3H]cholesterol by gamma-LpE by 40%. Totally, the cholesterol efflux capacity of plasma decreased by 10% compared to the original sample. The amount of immunodetectable pre beta 1-LpA-I increased when plasma was incubated in the presence of PLTP while the amount of immunodetectable gamma-LpE did not change. After one minute incubation of PLTP-conditioned plasma with [3H]cholesterol-labeled fibroblasts, the amount of radioactive cholesterol taken up by pre beta 1-LpA-I was twice as high as in control plasma whereas the amount of [3H]cholesterol taken up by gamma-LpE remained unchanged. As a net result, treatment with PLTP increased the cholesterol efflux into total plasma by 40%. Together with results of previous studies our data suggest that the conversion of alpha-LpA-I3 into alpha-LpA-I2 by PLTP generates pre beta 1-LpA-I but not gamma-LpE. PLTP helps to enhance the uptake of cell-derived cholesterol by pre beta 1-LpA-I and, thereby, the cholesterol efflux capacity of normal plasma.
Assuntos
Proteínas de Transporte/metabolismo , Lipoproteínas HDL/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Transferência de Fosfolipídeos , Apolipoproteína A-I/metabolismo , Apolipoproteínas E/metabolismo , Células Cultivadas , Colesterol/metabolismo , Eletroforese em Gel Bidimensional , Fibroblastos/metabolismo , Humanos , Immunoblotting , Lipoproteína(a)/análogos & derivados , Lipoproteína(a)/metabolismo , Doença de Tangier/metabolismoRESUMO
BACKGROUND: We determined whether the vascular effects of estradiol depend on the route of administration by comparing the effects of oral estradiol and transdermal placebo, transdermal estradiol and oral placebo, and transdermal placebo and oral placebo on in vivo endothelial function in 27 postmenopausal women. METHODS AND RESULTS: Endothelial function was assessed from blood flow responses to intrabrachial artery infusions of endothelium-dependent (7.5 and 15 microgram/min acetylcholine) and endothelium-independent (3 and 10 microgram/min of sodium nitroprusside) vasodilators at 0, 2, and 12 weeks. In the oral estradiol group, the increase in flow above basal during infusion of the low dose of acetylcholine at 0, 2, and 12 weeks averaged 6.0+/-0.8, 6.9+/-0.8, and 11.3+/-1.2 (P<0.01 versus 0 and 2 weeks) mL. dL(-1). min(-1) at 0, 2, and 12 weeks. The percentage increases versus 0 weeks averaged 21+/-14% at 2 and 120+/-34% at 12 weeks. During the high-dose acetylcholine infusion, the increase in flow above basal averaged 8.6+/-1.3, 10.2+/-1.5, and 15.1+/-1.8 (P<0.05 versus 0 weeks) mL. dL(-1). min(-1), respectively. The percentage increases versus 0 weeks averaged 22+/-10% at 2 weeks and 119+/-46% at 12 weeks. In the oral estradiol group, endothelium-independent vasodilatation also improved significantly, but less markedly than endothelium-dependent responses. In the transdermal and placebo groups, all vascular responses remained unchanged. Oral but not transdermal estradiol also induced significant decreases in LDL cholesterol and Lp(a) concentrations and an increase in HDL cholesterol within 2 weeks. CONCLUSIONS: We conclude that oral but not transdermal estradiol induces potentially antiatherogenic changes in in vivo endothelium-dependent vasodilatation and lipid concentrations.