P.S.M. Caines

A simple micromethod for rapid assessment of the distribution of apolipoprotein C isoforms in very-low-density lipoprotein

A simple and rapid isoelectric focusing method for quantifying Apo C isoforms of triglyceride-rich lipoprotein was developed. The very-low-density lipoprotein (VLDL) was isolated from 100 microL of EDTA plasma using a Beckman Airfuge ultracentrifuge. The delipidated VLDL was applied to an ultrathin flat acrylamide gel, and focused using a Bio-Rad Mini IEF Cell, for 1.5 h at a maximum of 500 V. Apo CII and Apo CIII in VLDL were resolved into four major bands, CIII0 (PI 4.91), CII (PI 4.78), CIII1 (PI 4.72), and CIII2 (PI 4.53). The method demonstrated within-run and between-run CVs of 2.7% to 11.9% and 4.4% to 12.2%, respectively. The relative percentage of C apoproteins and the ratio of CII to CIII found in VLDL from plasma of normal, chronic renal failure, and hyperlipidemic subjects agreed with previously published data.

A fluorometric coupled enzymatic method for the determination of adenosine triphosphate in platelets

Abstract A fluorometric coupled enzymatic method for the determination of adenosine triphosphate is described. The kinetic method can determine adenosine triphosphate in levels as low as 0.3 pmol. Intra- and interassay coefficients of variation were both less than 3% and recoveries were found to be quantitative. No significant interference was observed from EDTA or heparin. Correlation with an established method was significant ( r = 0.99). Clinical studies of the applicability of the procedure were demonstrated by the measurement of adenosine triphosphate in platelets. Mean adenosine triphosphate levels in platelets of “normal” subjects were found to be 2.17 ± 0.76 nmol/10 8 platelets or 14.22 ± 6.32 nmol/ mg platelet protein.

Determination of non-enzymatically glycated apolipoprotein A1

A procedure for the quantitation of non-enzymatically glycated apolipoprotein A1 (GApoA1) was developed and optimized. Glycated total protein was separated from plasma using m-aminophenyl-boronate affinity chromatography. Apolipoprotein A1 present in the glycated and non-glycated fractions of each sample was determined by rate nephelometry, and the percent glycated apo A1 calculated. The measuring range of the assay was 0.5-8.0% GApoA1. The within- and between-run CVs were less than 5.2 and 7.9%, respectively, and recoveries were greater than 92%. Free glucose did not affect the results. In a group of female non-insulin diabetic subjects the mean GApoA1 was 3.8 +/- 1.6% (mean +/- SD). In non-diabetic subjects the mean level of GApoA1 was 2.1 +/- 0.8% (mean +/- SD).

A competitive chemiluminescent immunoassay for the sensitive measurement of apolipoprotein B100

OBJECTIVES We have developed a competitive chemiluminescent immunoassay for the sensitive measurement of apolipoprotein B100 (apoB) and conducted a preliminary evaluation of the method. DESIGN AND METHODS In the assay, apoB-rabbit immunoglobulin G (IgG) conjugate competes with apoB in the sample for binding to acridinium N-hydroxysuccinimide labelled anti-apoB antibody. Goat anti-rabbit-IgG immobilized to magnetic particles is used to separate the apoB-rabbit IgG conjugate bound to the labelled anti-apoB antibody. Chemiluminescence, measured using a Ciba Corning Magic-Lite chemiluminometer, is inversely proportional to the concentration of apoB in the sample. RESULTS The assay is completed in 1-2 h. Within-run imprecision (n = 5) determined at 10, 100, and 200 micrograms/L of apoB was found to be 4.8%, 2.4%, and 3.5%, respectively. The between-run imprecision (n = 5) at the same concentrations of apoB was determined to be 14.2%, 9.5%, and 7.7%, respectively. The proposed assay showed reasonable correlation (r = 0.86) with a commercially available immunoturbidimetric method. The lower limit of detection was 2.2 micrograms/L (4.0 pmol/L). CONCLUSIONS This assay may be useful in tissue culture studies where sensitive measurement of secreted and intracellular concentrations of apoB are required. Our preliminary evaluation of the assay appears to confirm is usefulness.

A simple micromethod for rapid phenotyping of apolipoprotein E

Abstract Polymorphysim of apolipoprotein E (Apo E) accounts for a substantial amount of the genetically determined variance of cholesterol levels in the population, and is an important factor in the development of type III hyperlipidemia. In this report, we describe a simple and rapid isoelectric focusing method for phenotyping apolipoprotein E of triglyceride-rich lipoprotein. The very-low-density lipoprotein (VLDL) was isolated within 3 h from 100 μl of EDTA plasma using a Beckman Airfuge ultracentrifuge. The delipidated VLDL was applied to an ultrathin flat acrylamide gel, and focused using a Bio-Rad Mini IEF Cell, for 1.5 h at a maximum of 500 V. Apo E was resolved into three major bands, Apo E4 (pI 5.98 ± 0.02), Apo E3 (pI 5.83 ± 0.03), and Apo E2 (pI 5.71 ± 0.02). These bands were identified by cysteamine treatment and by immunofixation isoelectric focusing using goat antibody to Apo E. The present method can be used in routine clinical laboratories and can be performed in 1 day.