Effects of Cytokines, Butyrate and Dexamethasone on Serum Amyloid A and Apolipoprotein A-I Synthesis in Human HUH-7 Hepatoma Cells

Serum amyloid A (SAA) and apolipoprotein A-I (apo A-I) are secreted by the liver. As concentrations of both apolipoproteins are inversely related under normal and acute-phase conditions, human HUH-7 hepatoma cells were stimulated with interleukin (IL)-1alpha (100 and 200 U), IL-6 (50 and 100 U), butyrate (2 mM) and dexamethasone (2 x 10(-7)M and 1 x 10(-6)M), alone or in combination. Changes in SAA and apo A-I synthesis were monitored after metabolic labelling of the cells with [35S]-methionine. Intracellular and secreted SAA and apo A-I were immunoprecipitated, separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and the radioactivity in the corresponding bands was counted. Intracellular apolipoprotein levels were increased by all stimuli, either alone or in combination, between 2.7- and 5.5-fold (SAA) and between 2.8- and 4.1-fold (apo A-I), respectively. In a similar manner, apolipoprotein levels secreted by HUH-7 cells were increased between 3.1- and 4.3-fold (SAA) and between 1.9- and 3. 3-fold (apo A-I). Co-administration of cytokines, butyrate and/or dexamethasone had no pronounced synergistic effect on intracellular biosynthesis and secretion of SAA and apo A-I. The results from the present study suggest that apo A-I must not necessarily be considered as a negative acute-phase reactant.     

High-Density Lipoprotein has Different Binding Capacity for Different Apoproteins

Purified human amyloid protein A (AA) or serum amyloid protein A (SAA) was incubated with normal human high-density lipoprotein (HDL). After ultracentrifugation the amount of AA or SAA associated with HDL was measured. It was found that the binding capacity of HDL for SAA was higher than that for AA. Incubation of these in vitro associated HDL-AA and HDL-SAA complexes with purified apo AI or apo AII resulted in varying degrees of displacement of the associated AA or SAA from HDL. Under the experimental conditions used, apo AI was able to displace AA from HDL, while apo AII was able to displace both SAA and AA. This indicates that the binding capacity of HDL is different for SAA and AA. Mouse acute-phase HDL was isolated and the native complexes were incubated with human apo AII. SAA2, the amyloidogenic SAA variant in mice, was displaced from HDL to a greater extent than SAA1, indicating a lower binding capacity for the amyloidogenic SAA variant for the HDL complexes.     

Serum amyloid A apolipoprotein (apo SAA). Implications in inflammation and in lipoprotein modifications

The measurement of serum amyloid A apolipoprotein (apo SAA) during acute phase inflammation offers a high interest because of its specificity, sensitivity and early increase of its levels, compared to other acute phase proteins. Furthermore apo SAA is transported in serum in association with lipoproteins, in particular with their denser subpopulation, HDL3 thus inducing their modification. The decrease in Lp AI:AII concentrations in inflammatory diseases is the consequence of the decrease in HDL3. In general the HDL3 composition was changed with a displacement of apo AI by SAA. Another interest to this protein is its relationship with amyloidosis. Apo SAA is the presumed precursor of amyloid A protein, which can be deposited in various tissues, leading to secondary amyloidosis.     

First Evidence for the Existence of Multiple Isoforms of Bovine Serum Amyloid-A (apoSAA)

Bovine serum amyloid-A (SAA) was purified from acute-phase high density iipoprotein (HDL) by affinity chromatography and subsequent gel filtration chromatography. The identity of the isolated protein was checked by Western blotting following SDS-urea-PAGE using antisera raised against the purified protein fraction (SAA) and Amyloid A (AA). The antiserum raised against the purified SAA stained Congo red positive regions in the kidney of an AA-amyloidotic cow and reacted on Western blot with an AA-related protein of approximately 14kDa. Moreover, it immunostained two to three bands, of approximately 14kDa, present in serum from diseased cows, proportionally to the serum SAA concentration as measured by ELISA.
Isoelectric focusing of the purified bovine SAA fraction revealed three major (pI 5.5, 6.0, 6.4) and three minor (pi 4.8, 5.0, 7.3) isoforms and two-dimensional SDS-urea-PAGE confirmed the identity of the major isoforms. Isoelectric focusing of SAA isolated from sera, obtained from cows affected with different diseases, showed a variable ratio of the isoforms. In SAA isolated from serum obtained from acow suffering from spontaneous AA-amyloidosis only one isoform (pI 4.8) was detectable.
It is concluded that the results give first evidence for the existence of multiple isoforms of bovine SAA, occurring in different plasma concentration ratios during different diseases.     

Ⅳ型高脂血症患者高密度脂蛋白亚类组成与载脂蛋白E基因多态性关系的研究

目的探讨型高脂血症患者载脂蛋白E基因多态性与HDL亚类组成的关系。方法采用聚合酶链反应-限制性片段长度多态性和双向电泳-免疫印迹检测法,分析103例型高脂血症患者和146名血脂正常者的apoE基因型、HDL各亚类组成及相对含量。结果型高脂血症组和对照组apoE基因型及等位基因频率分布均以E3/3和ε3最高。型高脂血症患者中等位基因ε2携带者血清HDL-C、apoE、apoE/C、HDL2a较等位基因ε3、ε4携带者升高,而TG/HDL-C、apoC则下降,等位基因ε2携带者HDL3c较等位基因ε3携带者降低,其差异有统计学意义(P<0.05)。对照组中等位基因ε2携带者血清TG、apoE、apoE/C较等位基因ε3和ε4携带者升高,等位基因ε2携带者HDL3a较等位基因ε3携带者降低,其差异有统计学意义(P<0.05)。结论型高脂血症患者apoEε2等位基因与血清HDL亚类的成熟代谢有关。     

Characterization of Amyloid Proteins AA and SAA as Apolipoproteins of High Density Lipoprotein (HDL)

An AA-like protein with a molecular weight of 8600 complexed to high-density lipoprotein (HDL) was demonstrated in several acute-phase sera with high levels of SAA. The protein 'apo AA' (to distinguish it from tissue AA) was isolated by elution from sodium dodecyl sulphate (SDS)-polyacrylamide gel, and showed antigenic identity with purified tissue protein AA in double immunodiffusion. Normal HDL was shown to bind purified tissue AA in vitro. When the in vitro-associated HDL-AA complexes were given intravenously to mice during induction of amyloidosis, human AA was incorporated in the amyloid fibrils. Both apo AI and apo AII were shown to displace SAA from acute phase HDL when added to HDL-SAA complexes in vitro. This might be of importance in amyloidogenesis, as the liver and the small intestine, which are the main sites for AI and AII synthesis, are also sites of early amyloid deposition.     

Is the serum amyloid A protein in acute phase plasma high density lipoprotein the precursor of AA amyloid fibrils?

Serum amyloid A protein (SAA), an apolipoprotein of high density lipoprotein (HDL), is generally considered to be the precursor of AA protein, which forms the fibrils in reactive systemic amyloidosis in man and animals. This view is based on amino acid sequence identity between AA and the amino-terminal portion of SAA. However, in extensive and well-controlled studies of experimentally induced murine AA amyloidosis, we were unable to demonstrate a direct precursor-product relationship between SAA, in SAA-rich HDL preparations from acute phase or amyloidotic mouse or human serum, and AA protein in the amyloid deposits. This raises the possibility that SAA in its usual form, as an apolipoprotein of HDL synthesized during the acute phase response, may not be the major precursor of AA fibrils. The amyloidogenic forms of circulating SAA molecules may not be isolated during the preparation of HDL. Alternatively, particularly in the light of recent evidence that SAA mRNA is expressed in many different tissues throughout the body of appropriately stimulated animals, amyloidogenic SAA may be derived from sources other than the liver cells in which SAA-rich HDL is synthesized.     

Both acute phase and constitutive serum amyloid A are present in atherosclerotic lesions

The polymorphic protein, serum amyloid A (SAA), consists of acute phase Isotypes and a constitutive Isotype. Both are associated mostly with high density lipoproteins (HDL) In the circulation. In the present study, both SAA isotypes were detected by Immunohistochemistry and Immunoblotting using monocional antlbodies In atherosclerotic lesions. As the distribution of SAA was identical with that of apolipoprotein B and SAA is known to be associated also with low density lipoproteins (LDL), SAA may also be delivered to the artery wall by LDL.     

肥胖者HDL亚类组成与载脂蛋白E基因多态性的关系

目的：探讨肥胖者血清载脂蛋白E基因多态性与HDL亚类组成的关系。 方法： 采用聚合酶链反应-限制性片段长度多态性和双向电泳-免疫印迹检测法，分析93例肥胖者和96例非肥胖者者的apoE基因型、HDL各亚类组成及相对含量。 结果： 肥胖组和对照组apoE基因型及等位基因频率分布均以E3/3和ε3最高。肥胖者等位基因ε2携带者血清apoE/CⅢ、HDL2a较等位基因ε3和ε4携带者升高，而apoB100、apoCIII、HDL3c则较ε3携带者下降，差异显著（P<0.05）。对照组中等位基因ε2携带者血清TC、apoE较等位基因ε3携带者升高，等位基因ε2携带者HDL3b较等位基因ε3携带者降低，差异显著（P<0.05）。 结论： apoE 基因多态性与HDL亚类的组成和分布相关,ε2等位基因有减缓肥胖者HDL颗粒变小的作用。     

血清载脂蛋白CⅡ含量对HDL亚类分布的影响

目的:探讨血清载脂蛋白CⅡ含量对HDL亚类分布的影响。方法:采用双向电泳-免疫印迹方法检测247例受试者血清HDL亚类组成及含量。结果:随血清apoCⅡ水平的逐渐升高,年龄、BMI、TG、TC、apoB100、apoCⅡ、apoCⅢ、apoE、preβ1-HDL、preβ2-HDL、HDL3b和HDL3a含量显著增加,HDL-C、HDL2a、HDL2b含量显著减少。随apoCⅡ和apoAⅠ水平的逐渐升高,preβ1-HDL的含量均呈增加趋势,但HDL2b的含量在同一apoCⅡ组随apoAⅠ水平的升高呈增加趋势,而在同一apoAⅠ组随apoCⅡ水平的增加呈减少趋势。随apoCⅡ/apoCⅢ比值的逐渐升高,preβ1-HDL的含量呈增加趋势,而HDL2b的含量呈减少趋势。相关分析发现,控制血清TG和TC浓度后,apoCⅡ与preβ1-HDL呈显著正相关(r=0.186,P0.01),与HDL2b呈显著负相关(r=-0.149,P0.05);apoAⅠ含量与所有HDL亚类呈显著正相关(r值在0.349-0.587之间,P0.01);此外,apoCⅢ与preβ1-HDL和preβ2-HDL呈显著正相关(r分别为0.184和0.178,P0.01);apoB100与HDL2a呈显著负相关(r=-0.102,P0.05);apoE与HDL3a呈显著正相关(r=0.040,P0.05)。结论:随血清apoCⅡ水平的逐渐升高,HDL亚类的颗粒呈减小趋势,提示HDL成熟代谢受阻;apoAⅠ的含量可以对抗apoCⅡ对HDL亚类分布的影响;apoCⅡ/apoCⅢ的比值亦可作为反映HDL亚类分布的指标之一。     

Serum Amyloid A Protein in Mink During Endotoxin Induced Inflammation and Amyloidogenesis

Two-dimensional electrophoresis was used to study SAA and AA proteins in mink during lipopoly-saccharide-induced inflammation and amyloidogenesis. Three isotypes, SAA pI 6.8 and SAA pI 6.5 (both SAA1-like), and SAA pI 6.0 (SAA1- and SAA2-like), were identifled in serum after both single and multiple LPS injections. Total SAA serum levels were highest in the early phase of induction, followed by a decrease ranging from 1 to 50% of the peak value during the rest of the experiment. The variation in the total SAA levels correlated with the total SAA mRNA levels. Low total SAA levels were seen both in non-amyloidotic and amyloidotic animals, and a general decrease of all isotypes was demonstrated. In hepatic amyloid fibrils, several AA isotypes, with amino acid sequence homologous exclusively to that of SAA2, were found. In the corresponding splenic material, fragments of histones H2A and H2B constituted most of the low molecular mass proteins, and no protein AA was detected. In spite of low serum levels and a non-specific isotype removal, the results confirm that SAA2 is amyloidogenie in mink.     

高脂血症APOE基因多态性与高密度脂蛋白亚类组成的关系

目的探讨高脂血症患者载脂蛋白E(apolipoproteinE,APOE)基因多态性与高密度脂蛋白(highdensitylipoprotein,HDL)亚类组成变化的关系。方法应用聚合酶链反应-限制性片段长度多态性和双向电泳-免疫印迹检测法,分析112例高脂血症患者和73名正常对照者APOE基因型、HDL各亚类组成及相对含量。结果高脂血症组和对照组APOE基因型及等位基因频率分布均以E3/3和ε3最高。高脂血症患者中等位基因ε2携带者血清APOE/C较等位基因ε3升高,而HDL3b则下降,其差异有显著性(P<0.05)。对照组中等位基因ε2携带者血清甘油三酯、apoE、apoE/C较等位基因ε3和ε4携带者升高,等位基因ε2携带者HDL3a较等位基因ε3携带者低,其差异有显著性(P<0.05)。结论APOE基因多态性可能与血清HDL部分亚类的含量变化相关。     

High-Density Lipoprotein as Carrier for Amyloid-related Protein SAA in Rabbit Serum

In this study present evidence that SAA is complexed to high density lipoprotein (HDL) in rabbit serum and is co-isolated with HDL apoproteins. The binding of SAA to HDL seems to be quite strong, judged from affinity chromatography experiments. The studies did not reveal any interaction between SAA and albumin, and there was no evidence that SAA could complex to itself. By isolation of HDL apoproteins, SAA seems to behave like other known apoproteins and may be characterized as an apoprotein that is present in normal serum in very low concentration but increases in concentration under different unphysiological circumstances.     

Endogenous apolipoprotein E modulates cholesterol efflux and cholesteryl ester hydrolysis mediated by high-density lipoprotein-3 and lipid-free apolipoproteins in mouse peritoneal macrophages

We investigated the effect of endogenous apolipoprotein (apo) E synthesis in mouse peritoneal macrophages on cholesterol efflux and intracellular cholesteryl ester hydrolysis mediated by high-density lipoprotein-3 (HDL3) and lipid-free apolipoproteins (apo). After loading with acetylated LDL (acLDL) peritoneal macrophages from wild-type (apoE(+/+)) and apoE-deficient (apoE(-/-)) mice were incubated with medium alone or with liposomes, HDL3, lipid-free apoA-I, or lipid-free apoE3. Cholesterol and cholesteryl esters in the cells and culture media were quantified by HPLC. Incubation of apoE(+/+) or apoE(-/-) macrophages for 18 h with medium alone or with liposomes did not cause significant changes in cellular cholesterol. Addition of HDL3, apoA-I, or apoE3 to the medium led to significant cholesterol efflux, which was less efficient in apoE(-/-) macrophages than in apoE(+/+) macrophages. HDL and lipid-free apolipoproteins were more effective in reducing the cellular content of cholesteryl esters of apoE(+/+) macrophages than of apoE(-/-) macrophages, suggesting that endogenous apoE stimulates cholesteryl ester hydrolysis. The difference in the mass of cholesteryl esters was more pronounced for cholesteryl arachidonate and linoleate than for cholesteryl oleate or palmitate. Furthermore, in [(14)C]arachidonate labeling experiments cholesterol arachidonate hydrolysis was higher in apoE(+/+) macrophages than in apoE(-/-) macrophages in the presence of cholesterol efflux mediated by HDL3 or apoA-I. In contrast, in the absence of cholesterol efflux cholesterol arachidonate synthesis was higher in apoE(+/+) macrophages than in apoE-/- macrophages. Taken together, our data suggest that endogenous apoE stimulates cholesterol efflux and intracellular cholesteryl ester hydrolysis mediated by HDL3 and lipid-free apolipoproteins in mouse peritoneal macrophages. This may contribute to the antiatherogenic effect of apoE.     

Characterization of a monoclonal antibody that binds to apolipoprotein E and to lipoprotein of human plasma containing apo E. Applications to ELISA quantification of plasma apo E

This study describes the development of an enzyme-linked immunosorbent assay for human apolipoprotein E (apo E). A mouse monoclonal IgG1 antibody named E01 against apolipoprotein E was selected from five antibodies secreted by hybridomas. This antibody had a high affinity for apo E [K = 1.2 x 10(7) L.M-1 for purified apo E and K = 1.05 x 10(7) L.M-1 for native apo E in very low density lipoproteins) in liquid phase and recognized every isoform of apo E but not other proteins in VLDL. Competition experiments with 125I apo E showed that its binding affinity for the E in every density class (VLDL, HDL, LDL) and in serum was the same. This antibody was used for the quantification of human apo E in serum by enzyme linked immunoassay. E01 was coated on microtiter plates and a polyclonal peroxidase-conjugate was used as second antibody. A good correlation was observed between the values obtained for apo E using both monoclonal and polyclonal antibodies.     

Metabolic disorders of lipoproteins--influences of compositional changes of lipoproteins upon their metabolic behavior

Compositional changes of apoproteins and lipids in lipoproteins influence their affinities for receptors and enzymes. Decrease of apo C proteins and increase of apo E in chylomicron and very low density lipoproteins (VLDL) during their catabolism might promote the binding to remnant receptor. On the other hand, the affinity for lipoprotein lipase (LPL) gradually decreases and that for hepatic lipase increases. However, the responsiveness of VLDL to LPL might be under the control of triglyceride (TG)/surface component ratios but not of the apoprotein ratios in ordinary circumstances judging from the results of the releases of fatty acids from VLDL by LPL in vitro. Responses of VLDL from diabetic patients to LPL significantly decreased compared with those from non-diabetic subjects. Glycation of VLDL in vitro impaired their responses to LPL. Therefore, delayed catabolism of VLDL in diabetes might partially depend upon glycation of VLDL besides the decreased LPL activity. Low density lipoproteins (LDL), apoproteins of which consist mostly of apo B protein and had a low TG level, showed a high affinity to the LDL receptor. However, LDL from hypertriglyceridemic subjects, in which the TG contents was increased, had a low affinity to the receptor. Since high density lipoproteins (HDL) from patients in acute phases contain a large amount of serum amyloid A protein (SAA), the percentages of apo A proteins markedly decreased. When SAA-rich HDL were incubated with leucocytes, SAA were degraded rapidly, although other apoproteins remained to be unchanged. Therefore, such HDL become unstable, and this might induce low HDL levels in the acute phase.     

高脂血症高密度脂蛋白亚类分布与Apo A-Ⅰ基因多态性的关系

目的：探讨高脂血症患者高密度脂蛋白（HDL）亚类分布与载脂蛋白A-Ⅰ(Apo A-Ⅰ)基因多态性的关系。 方法： 采用聚合酶链反应(PCR)-限制性片段长度多态性(RFLP)和双向电泳-免疫印迹检测法，分析比较118例高脂血症患者和109例血脂正常者的Apo A-Ⅰ基因型、HDL各亚类分布及相对含量。 结果： Apo A-Ⅰ基因 －78 bp 位点和 ＋83 bp 位点的多态性分别以G/G和C/C基因型占优势，其中 －78 bp 位点高脂血症组A等位基因的频率显著高于对照组（P＜0.05）。高脂血症患者中，G/A突变受试者血清TG、Apo C-Ⅲ、pre β1-HDL及HDL3a水平显著高于，而HDL2a和HDL2b水平则显著低于G/G基因型者。 结论： 高脂血症Apo A-Ⅰ基因 －78 bp 位点G/A突变与HDL亚类分布相关，G/A突变受试者血清HDL亚类颗粒呈减小的趋势，提示HDL成熟代谢可能受阻。     

A case of hyper-HDL-cholesterolemia presenting peculiar lipoprotein patterns in agarose gel electrophoresis

Lipoprotein metabolism was analyzed in a patient with marked hyper-HDL-cholesterolemia. A 50 year old male with no symptom of ischemic heart disease or xanthoma had a serum cholesterol level between 293 and 410 mg/dl, and a markedly elevated, HDL-cholesterol level (160-190 mg/dl). The cholesterol content of ultracentrifugally separated HDL2 was exclusively increased, while it was normal in the HDL3 fraction. Analytical ultracentrifugation and HPLC revealed that HDL particles became remarkably larger than the control and, on the contrary, LDL particles became smaller. LPL and LCAT activities were higher in this case, but H-TGL activity was normal. Agarose gel electrophoresis of lipoproteins showed an abnormal broad band which was located between alpha and pre beta band. Serum levels of apolipoprotein A-I, A-II, C-II, C-III and E were higher, while apolipoprotein B level was slightly lower than the control. Cholesteryl ester transfer protein (CETP) activity was demonstrated to be completely deficient in this case, as determined in 10 microliters serum using [3H] CE-labeled HDL3 as donor and VLDL + LDL fraction as acceptor. Since CETP was considered to catalyze the cholesteryl ester transport from HDL to VLDL and LDL, the deficiency of this activity might be the cause of the marked hyper-HDL-cholesterolemia in this patient.     

高脂血症患者HDL亚类组成与脂蛋白酯酶基因内含子8 HindⅢ多态性的关系

目的：探讨高脂血症患者脂蛋白酯酶基因内含子8 HindⅢ酶切位点多态性与HDL亚类组成的变化关系。 方法： 采用聚合酶链反应-限制性片段长度多态性和双向电泳-免疫印迹检测法，分析152例高脂血症患者和128例血脂正常者的脂蛋白酯酶基因内含子8 HindⅢ多态性、HDL亚类组成及相对百分含量。 结果： 高脂血症组和对照组均以H+H+基因型为主，但高脂血症组H+H+基因型频率显著高于对照组，而H+H-和H-H-基因型频率显著低于对照组（P<0.05）。高脂血症组H+等位基因频率显著高于对照组，而H-等位基因频率显著低于对照组（P<0.05）。高脂血症组和正常对照组H+H+基因型者血清TG明显高于H-H-者；高脂血症组H+H+基因型者apoB100、TG/HDL-C比值明显高于H-H-者（P<0.05）。高脂血症患者H+H+基因型小颗粒的preβ1-HDL、HDL3b相对含量多于H-H-者，大颗粒的HDL2a、HDL2b相对含量少于H-H-者；正常对照组H+H+基因型HDL3c多于H-H-者，而HDL2a少于H-H-者，其差异均显著（P<0.05）。 结论： 中国人高脂血症患者脂蛋白酯酶基因HindⅢ酶切位点多态性与HDL亚类的组成相关。     

Lipopolysaccharide is transferred from high-density to low-density lipoproteins by lipopolysaccharide-binding protein and phospholipid transfer protein

Lipopolysaccharide (LPS), the major outer membrane component of gram-negative bacteria, is a potent endotoxin that triggers cytokine-mediated systemic inflammatory responses in the host. Plasma lipoproteins are capable of LPS sequestration, thereby attenuating the host response to infection, but ensuing dyslipidemia severely compromises this host defense mechanism. We have recently reported that Escherichia coli J5 and Re595 LPS chemotypes that contain relatively short O-antigen polysaccharide side chains are efficiently redistributed from high-density lipoproteins (HDL) to other lipoprotein subclasses in normal human whole blood (ex vivo). In this study, we examined the role of the acute-phase proteins LPS-binding protein (LBP) and phospholipid transfer protein (PLTP) in this process. By the use of isolated HDL containing fluorescent J5 LPS, the redistribution of endotoxin among the major lipoprotein subclasses in a model system was determined by gel permeation chromatography. The kinetics of LPS and lipid particle interactions were determined by using Biacore analysis. LBP and PLTP were found to transfer LPS from HDL predominantly to low-density lipoproteins (LDL), in a time- and dose-dependent manner, to induce remodeling of HDL into two subpopulations as a consequence of the LPS transfer and to enhance the steady-state association of LDL with HDL in a dose-dependent fashion. The presence of LPS on HDL further enhanced LBP-dependent interactions of LDL with HDL and increased the stability of the HDL-LDL complexes. We postulate that HDL remodeling induced by LBP- and PLTP-mediated LPS transfer may contribute to the plasma lipoprotein dyslipidemia characteristic of the acute-phase response to infection.