Characterization and quantification of serum lipoprotein subfractions by capillary isotachophoresis: relationships with lipid, apolipoprotein, and lipoprotein levels.

Human serum lipoproteins are currently defined according to their density as well as according to their electrophoretic mobility. They can be fractionated into discrete subspecies which exhibit variations in their structure and function. Capillary electrophoresis has been suggested to be a potential analytical strategy in understanding metabolic lipoprotein heterogeneity. In a sample of 35 normolipidemic subjects, we analyzed ceramide-labeled serum lipoproteins by capillary isotachophoresis linked to laser-induced fluorescent detection. Capillary isotachophoresis showed advantage to be an automated, rapid (6 min) and reproducible (CV < 7%) separation mode, on-line monitoring lipoprotein subfractions according to net charge. HDL were separated into three subfractions: i) the fast migrating HDL correlated positively with serum apoA-I (P < 0.05) and negatively with triglyceride (P < 0.01) concentrations, ii) the intermediate migrating HDL involved in HDL-cholesterol delivery and inversely related to LDL particles concentration (P < 0.001), and iii) the slow migrating prebeta(1)HDL. Triglyceride level was significantly associated with two fractions: i) the VLDL fraction correlated positively with apoE serum concentration (P < 0.01), and ii) the IDL fraction closely and positively associated with apoC-III-containing lipoprotein level (P < 0.001). Two LDL subfractions were positively related to LDL-cholesterol (0.05

Capillary electrophoretic analysis of recombinant human apolipoprotein E. Calibration mode of a protein reference material.

Actual lack of standardization of serum apolipoprotein (apo) E measurements prevents intensive studies on the significance of apoE concentration in clinical chemistry. For this purpose the use of standards calibrated against a common reference material is essential in order to obtain reliable results. The assignment of a certified value to this reference material involves the use of an accurate and non immunological technique. We demonstrate here that capillary electrophoresis in sodium dodecyl sulfate containing gel (SDS-CGE) can be a method of choice. ApoE quantification was performed according to the peak area and a standard solution of purified apoAI. We obtained very close results to that obtained by HPLC of phenylalanine apoE content measurement i.e., 81.6 mg/l versus 84.6 mg/l and concluded that the SDS-CGE analysis is an accurate and reliable method for the certification of an apoE reference material to be used in serum apoE concentration measurements. The SDS-CGE combines advantages of SDS-polyacrylamide gel electrophoresis (high resolving power, rapidity and tolerance of complex sample) with that of quantitative HPLC analysis (accuracy, precision, linearity and speed).

DNA extraction and stability for epidemiological studies.

During the last few years, an important development of molecular biology techniques has been observed in research and clinical laboratories, and consequently the availability of DNA is becoming essential for epidemiological studies. Several DNA extraction procedures have been proposed. However as the quantity and quality of the DNA extracted is very variable, standardization of the storage of samples, and of extraction procedures becomes essential. Three steps will be considered. (i) Procedures of whole blood preservation prior to extraction which seem to affect yield and purity of the DNA extracted; (ii) DNA extraction procedures, which must be validated by a systematic DNA quality control using DNA molecular weight screening and spectrophotometric analysis, and also by verification that the DNA obtained is well adapted for molecular biology applications; (iii) the third important factor to study is the storage of DNA, since data on short-term (several months) and especially on long-term (several years) DNA stability is often missing and/or conflicting. Detailed studies must be done in order to establish guidelines for proper handling of genetic material.

High sensitivity of laser-induced fluorescence detection in capillary gel electrophoresis for accurate apolipoprotein E genotyping.

Human apolipoprotein E (apoE) is a product of a polymorphic gene. In the general population, it shows two major mutations, which lead to the appearance of three common alleles encoding for three protein isoforms. This polymorphism is important in the regulation of lipid metabolism. Accurate apoE phenotyping or genotyping has become essential in clinical laboratories, since the epsilon 4 allele has been associated with cardiovascular and Alzheimer's diseases. Endonuclease restriction isotyping, followed by slab gel electrophoresis, is a rapid and convenient method for the investigation of common apoE genotypes. However, during the large-scale apoE genotyping of the STANISLAS cohort, we were confronted with a partial lack of sensitivity and resolution power of this traditional method, which sometimes leads to the misclassification of the genotypes epsilon 2/2 and epsilon 3/2. We have overcome this difficulty by separating the restriction fragments with capillary gel electrophoresis linked to laser-induced fluorescence detection. The baseline resolution was 2 bp, and the sensitivity limit attainable was similar to that by radioactive detection. The distinction between the epsilon 3/2 and the epsilon 2/2 genotypes became unequivocal, even when only low amounts of DNA were available for amplification.

Apolipoprotein E genotype epsilon 4/epsilon 2 in the STANISLAS Cohort Study--dominance of the epsilon 2 allele?

Apolipoprotein (apo) E has been discussed as a marker for cardiovascular risk, but information about lipid traits in healthy individuals having one of the rare apoE genotypes (epsilon 4/epsilon 2, epsilon 2/epsilon 2 or epsilon 4/epsilon 4) is scarce. Our work was designed to answer the following questions: 1. Are the allelic effects of epsilon 2 and epsilon 4 on lipid traits additive or dominant? 2. If there is additivity, do the allelic effects of epsilon 2 and epsilon 4 have the same magnitude? 3. Are the allelic effects neutralised in epsilon 4/ epsilon 2 individuals who are under the influence of both rare alleles? Allelic effects on apoB and apoE serum levels were codominant. Allelic models are thus not adequate to study the influence of apoE polymorphism on these traits. Allelic effects were additive for total cholesterol, LDL-C, HDL-C and apoAI, with epsilon 2 having a greater impact than epsilon 4. Serum levels differed significantly between epsilon 4/epsilon 2 and epsilon 3/epsilon 3 individuals only for apoE (p < 0.001) and for apoB (p < 0.05).