In vivo kinetics of human apolipoprotein A-I variants in rabbits

BACKGROUND: Genetic variants of human apolipoprotein (apo) A-I, the major protein component of high-density lipoprotein (HDL), with a single amino acid substitution have been reported, and some of these result in very low plasma HDL-cholesterol (C) levels. Examining the kinetics of radiolabelled apolipoprotein is a straightforward technique for determining its metabolism in vivo. In this study, we investigated the in vivo kinetics of several human apo A-I variants, which we had identified previously, in rabbits. MATERIALS AND METHODS: Apo A-I variants from heterozygous carriers of Lys-107-->0, Lys-107-->Met, Pro-3-->Arg, Pro-4-->Arg, Pro-165-->Arg and Glu-198-->Lys and the corresponding normal apo A-I were purified and then radioiodinated with 131I and 125I. A kinetic study of apo A-I variants was performed in normolipidaemic rabbits after simultaneous injection of the two isotopes that had been incorporated into HDL. The fractional catabolic rate (FCR) was calculated from the radioactive decay curve. RESULTS: Acidic mature (negatively charged) apo A-I variants caused by a single amino acid substitution (Lys-107-->0, and Lys-107-->Met) were catabolized faster (FCR, 1.931 +/- 0.539 per day vs. 1.636 +/- 0.460 per day, P Arg, Pro-4-->Arg, Pro-165-->Arg and Glu-198-->Lys) were catabolized more slowly (FCR 1.470 +/- 0.380 per day vs. 1.654 +/- 0.430 per day, P 相似文献   

Apolipoprotein A-I variants. Naturally occurring substitutions of proline residues affect plasma concentration of apolipoprotein A-I.

Six unrelated families with genetically determined structural variants of apo A-I were found in the course of an electrophoretic screening program for apo A-I variants in dried blood samples of newborns. The following structural variations were identified by the combined use of HPLC, time-of-flight secondary ion mass spectrometry (TOF-SIMS), and automated gas phase sequencing: Pro3----Arg (1x), Pro4----Arg (1x), and Pro165----Arg (4x). All variant carriers were heterozygous for their mutant of apo A-I. Subjects heterozygous for apo A-I(Pro165----Arg) (n = 12) were found to exhibit lower mean values for apo A-I (109 +/- 16 mg/dl) and HDL cholesterol (37 +/- 9 mg/dl) than unaffected family members (n = 9): 176 +/- 41 and 64 +/- 18 mg/dl, respectively (P less than 0.001). In 9 of 12 apo A-I(Pro165----Arg) variant carriers the concentrations of apo A-I were below the fifth percentile of sex-matched controls. By two-dimensional immunoelectrophoresis as well as by densitometry the relative concentration of the variant apo A-I in heterozygous carriers of apo A-I(Pro165----Arg) was determined to account for only 30% of the total plasma apo A-I mass instead of the expected 50%. Thus, the observed apo A-I deficiency may be largely a consequence of the decreased concentration of the variant apo A-I. In the case of the apo A-I(Pro3----Arg) mutant, densitometry of HDL apolipoproteins demonstrated a distinctly increased concentration of the variant proapo A-I relative to normal proapo A-I. This phenomenon was not observed in the apo A-I(Pro4----Arg) mutant or in other mutants. This suggests that the interspecies conserved proline residue in position 3 of mature apo A-I is functionally important for the regular enzymatic conversion of proapo A-I to mature apo A-I.     

Sustained expression of human apolipoprotein A-I after adenoviral gene transfer in C57BL/6 mice: role of apolipoprotein A-I promoter, apolipoprotein A-I introns, and human apolipoprotein E enhancer

Elevation of HDL cholesterol, after adenoviral apolipoprotein A-I (apo A-I) gene transfer, may delay or revert ischemic cardiovascular disease, provided transgene expression is persistent. Previously, we observed transient human apo A-I expression after adenoviral gene transfer with a cytomegalovirus (CMV)-driven construct containing the human apo A-I cDNA. Therefore, the effects of promoters (CMV or 256 base pairs of the human apo A-I promoter), introns of the human apo A-I gene, and the liver-specific human apolipoprotein E (apo E) enhancer on adenovirus-mediated human apo A-I expression were evaluated in C57BL/6 mice. In the presence of the CMV promoter, human apo A-I introns prolonged expression above 20 mg/dl from 14 to 35 days. Addition of one, two, or four copies of the human apo E enhancer in these constructs resulted in a copy-dependent but transient increase in expression for 14 days. The apo A-I promoter induced 3.2-fold lower peak levels of human apo A-I than did the CMV promoter, but insertion of four apo E enhancers in the apo A-I promoter-driven construct resulted in human apo A-I levels above 20 mg/dl for 6 months. The decline between day 6 and day 35 of human apo A-I expression driven by the CMV promoter was due to (1) a 2.5-fold decline in transgene DNA levels that is not observed with apo A-I promoter-driven constructs, and (2) CMV promoter attenuation as evidenced by a 7.6-fold decline in the human apo A-I mRNA/human apo A-I DNA copy number ratio between day 6 and day 35. Hepatotoxicity, as evidenced by up to 10-fold higher serum levels of transaminases on day 6 after gene transfer with CMV promoter-driven constructs than with apo A-I promoter-driven constructs, probably caused the accelerated decline of transgene DNA. In conclusion, gene transfer with an adenovirus comprising the 256-bp apo A-I promoter, the genomic apo A-I DNA, and four apo E enhancers, all of human origin, is associated with low hepatotoxicity and with the absence of promoter shutoff resulting in human apo A-I expression above 20 mg/dl for up to 6 months.     

Standardization of apolipoprotein B and A-I measurements

Apolipoprotein B, the major protein of low-density lipoprotein, and apolipoprotein A-I, the major protein of high-density lipoprotein, can serve as important predictors of atherosclerotic cardiovascular disease. However, these apolipoprotein measurements have not realized their full potential because of inadequate standardization. Purified apolipoprotein A-I of known absolute mass, in lyophilized form, can serve as primary standard for apolipoprotein A-I, and freshly isolated low-density lipoprotein of narrow density range can serve as primary standard for apolipoprotein B. These primary standards can be used to assign target values to secondary reference material and calibrators by reference laboratories using standardized, validated immunoassay procedures. Lyophilized serum can serve as secondary reference material for apolipoprotein A-I. Freshly frozen serum pools should be used as reference material for apolipoprotein B until more practicable materials that do not exhibit matrix interactions are developed. Implementation of proper standardization procedures can lead to significant improvements in apolipoprotein measurements.     

Familial apolipoprotein E deficiency.

A unique kindred with premature cardiovascular disease, tubo-eruptive xanthomas, and type III hyperlipoproteinemia (HLP) associated with familial apolipoprotein (apo) E deficiency was examined. Homozygotes (n = 4) had marked increases in cholesterol-rich very low density lipoproteins (VLDL) and intermediate density lipoproteins (IDL), which could be effectively lowered with diet and medication (niacin, clofibrate). Homozygotes had only trace amounts of plasma apoE, and accumulations of apoB-48 and apoA-IV in VLDL, IDL, and low density lipoproteins. Radioiodinated VLDL apoB and apoE kinetic studies revealed that the homozygous proband had markedly retarded fractional catabolism of VLDL apoB-100, apoB-48 and plasma apoE, as well as an extremely low apoE synthesis rate as compared to normals. Obligate heterozygotes (n = 10) generally had normal plasma lipids and mean plasma apoE concentrations that were 42% of normal. The data indicate that homozygous familial apoE deficiency is a cause of type III HLP, is associated with markedly decreased apoE production, and that apoE is essential for the normal catabolism of triglyceride-rich lipoprotein constituents.     

Characterization of two monoclonal antibodies to human apolipoprotein A-I that also bind to rabbit apolipoprotein A-I. Application to ELISA evaluation of rabbit serum apo A-I

Two monoclonal antibodies against human apolipoprotein A-I were characterized to recognize rabbit apo A-I. By immunoblotting we determined that while neither antibody reacted with the other rabbit apolipoproteins they both recognized all rabbit apo A-I isoforms. Cotitration revealed that each antibody bound to different apo A-I epitopes. Then we developed a noncompetitive enzyme-linked immunosorbent assay (ELISA) to measure the concentration of total apolipoprotein A-I in rabbit serum. The ELISA curves of the different lipoprotein class and serum showed the similarity of the expression of the apo A-I epitopes recognized in each of them and in serum. This assay, as opposed to other techniques, offers several advantages such as sensitivity, specificity, and simplicity and avoids the use of radioisotope.     

Nephelometric assay of apolipoprotein A-I with a centrifugal analyzer

We developed an automated immunonephelometric assay for quantification of human apolipoprotein A-I (apo A-I) with a fluorescence light-scattering microcentrifugal analyzer. The presence of polyethylene glycol and Tween 20 in the reaction mixture ensures maximum exposure of the antigenic sites of the apoprotein so that immune complex formation occurs more rapidly (reaction is complete within 2 min) and to a greater extent. Lipemia and hemolysis do not interfere with the measurement of apo A-I. The method requires only 10 microL of specimen and is fast and easy to perform. Results vary linearly with apo A-I concentrations to 2.5 g/L. Assay precision (CV) was 3.1% for a specimen with an apo A-I concentration of 1.45 g/L, and the lower limit of detection was 0.15 g/L. Values for a candidate Reference Material agree well with those reported in an international survey (Clin Chem 1985;30:223-8).     

Frequency of apolipoprotein A-I mutants in the German population

A randomly chosen population in the area of Westphalia (West Germany) was screened for apolipoprotein A-I mutants. About 5000 individuals were investigated and compared with a group of 1300 patients who had undergone coronary angiography. Four electrophoretically different apolipoprotein A-I-mutants (named Münster-1 to 4) were discovered. Five non-related probands were observed in the group of the unselected patients and three non-related probands in the group of coronary angiography patients. In most cases the familial nature of the abnormality was confirmed by pedigree analysis.     

Serum prostacyclin stabilizing factor is identical to apolipoprotein A-I (Apo A-I). A novel function of Apo A-I.

Serum PGI2 stabilizing factor (PSF) was purified from human serum to a single protein with a molecular weight of 28,000 D by SDS-PAGE. Analyses of NH2-terminal sequence (32 residues), COOH-terminal sequence (3 residues) and the composition of amino acids disclosed its homology with human apolipoprotein A-I (Apo A-I), a major apolipoprotein of HDL. Apolipoprotein A-II, C-I, C-II, C-III, D and E, as well as LDL, and VLDL did not possess this activity. The alpha-helix structure of Apo A-I is necessary for the binding of PGI2. HDL and nascent HDL reconstituted from Apo A-I and phospholipid significantly prolonged the half-life of PGI2. PGI2 stabilization by HDL and Apo A-I may be an important protective action against the accumulation of platelet thrombi at sites of vascular damage. The beneficial effect of HDL in the prevention of coronary artery disease may be partly due to this action.     

In vivo metabolism of apolipoprotein S in humans. Comparison with apolipoprotein A-I metabolism

¹²⁵I-labelled apolipoprotein (Apo) S and ¹³¹I-labelled apolipoprotein A-I were injected i.v. into healthy volunteers. Blood samples and daily urine collections were drawn periodically for 15 days. Ninety-eight percent of ¹³¹I radioactivity and > 95% of ¹²⁵I radioactivity were found in HDL after Superose gel chromatography of plasma. About 10% of each radioactivity was recovered in the d 1.250 infranate after one ultracentrifugation. Affinity chromatography on monoclonal anti-Apo A-I Sepharose column separates two lipoprotein particles containing Apo S, one retained with Apo A-I (42.5%) and the other eluting without Apo A-I (57.5%).

Kinetic parameters were calculated according to exponential curve fitting. Mean transit time was about 7.0 days for both Apo A-I and Apo S. FCR of Apo S was 50% higher than FCR of Apo A-I. Synthetic rate of Apo S was about 150 times smaller than for Apo A-I.

As heterogeneity of HDL-S was suggested by both the results of affinity chromatography and the urinary data, a compartmental model was built which fitted adequately all data. Part of the model is common to HDL-A-I and HDL-S.     

Apolipoprotein A-I, apolipoprotein B, and apolipoprotein B/apolipoprotein A-I ratio: reference intervals compared with values in different pathophysiological conditions from the FINRISK 2007 study

BackgroundIn addition to traditional measurements of serum lipid levels, apolipoprotein A-I (apoA-I), apolipoprotein B (apoB), and apoB/apoA-I ratio may add more value to risk assessment guidelines for cardiovascular disease.MethodsWe calculated reference intervals for apoA-I, apoB, and apoB/apoA-I ratio using a reference sample (n = 2828) from the FINRISK 2007 study.ResultsThe reference intervals for apoA-I were 1.1–2.0 g/l for men and 1.2–2.3 g/l for women. The corresponding reference intervals for apoB were 0.6–1.5 g/l and 0.6–1.3 g/l. The reference intervals for apoB/apoA-I ratio were 0.3–1.0 for men and 0.3–0.8 for women. Compared with the healthy reference group, obese men had the lowest ApoA-I, the highest apoB, and the highest apoB/apoA-I ratio. Men with CVD and cholesterol-lowering medication, or diabetes had lower apoB levels and apoB/apoA-1 ratio than the reference group but the opposite was true for women. The therapeutic goal for low-risk individuals for apoB was 0.9 g/l coinciding with LDL-C concentration of 3.0 mmol/l.ConclusionsReference intervals for apoA-I, apoB, and the apoB/apoA-I ratio and their cutoff values may be useful for the risk evaluation and follow-up of treatment among individuals having CVD or other metabolic disorders.     

Quantitative determination of human plasma apolipoprotein A-I by laser immunonephelometry

A technical procedure is described for quantitation of human apolipoprotein A-I (apo A-I) in normal plasma or serum by immunonephelometry. Dilution of the plasma samples with 6 mol/L guanidine chloride ensures maximum exposure of the antigenic sites of the apoprotein and enables optimum quantitation of the apo A-I without requiring extraction with organic solvents. Similar data are obtained by this assay and with radioimmunoassay for normal subjects (1.2--1.5 g/L), and the results obtained on 31 patients are correlated with a coefficient of 0.92. The apo A-I values are correlated with values for plasma high-density lipoprotein cholesterol (r = 0.64). The interassay CV for immunonephelometry is about 7% and the standard curve is linear between 0.1 and 1.0 microgram of apo A-I per sample, corresponding to a 150-fold dilution of serum or plasma. The assay is applicable to plasma samples containing as much as 4 g of triglycerides per liter. At higher concentrations plasma delipidation is required.     

Differential turbidimetric assay for subpopulations of lipoproteins containing apolipoprotein A-I

A differential immunoturbidimetric procedure for the quantitation of apolipoprotein A-I associated with lipoproteins LpA (containing both apolipoprotein A-I and apolipoprotein A-II) and with lipoproteins LpA-I (containing apolipoprotein A-I but no apolipoprotein A-II) is presented. Lipoproteins containing apolipoprotein A-II are precipitated with an anti-apolipoprotein A-II antibody. The resulting immunoprecipitate is sedimented and LpA-IA-I is measured in the supernate. Whereas LpA-IA-I concentrations differed significantly between normolipidaemic men and women (0.75 and 1.00 g/l, respectively), there was virtually no sex related difference in LpAA-I (0.83 and 0.88 g/l, respectively). LpA-IA-I was predominantly correlated with HDL2-cholesterol (rs = 0.630), whereas LpAA-I was statistically associated with HDL3 (rs = 0.417).     

载脂蛋白A1水平在不同脊髓损伤患者中的差异性研究

目的:通过对不同脊髓损伤患者血清载脂蛋白A1(ApoA-I)水平回顾性资料的流行病学调查,进行差异比较和相关因素分析,探讨造成这一心血管保护因子差异形成的可能机制和临床意义。方法:对中国康复研究中心2004年1月—2010年12月因脊髓损伤入院进行康复治疗的患者进行入院后ApoA-I水平回顾性调查。结果:女性脊髓损伤患者ApoA-I水平高于男性患者,截瘫患者ApoA-I水平高于四肢瘫患者,男性患者ApoA-I水平还受年龄和病程影响,女性患者ApoA-I水平与年龄和病程不相关。结论:ApoA-I水平在不同脊髓损伤类型患者间存在差异,男女不同性别之间也存在差异,雌激素保护作用和运动能力不同可能是造成差异的主要原因。