Abstract
The procedure for the measurement of serum apoprotein A-IV by rocket immunoelectrophoresis is described. Very high density lipoprotein (VHDL) was isolated from fresh pooled human plasma by ultracentrifugation. The VHDL was dialyzed against saline and incubated with a quarter volume of IntralipidR at 37°C for 1 hour. The incubation mixture formed semisolid creamy layers after a 16-hour ultracentrifugation at 15°C. Proteins incorporated to lipids particles were obtained by delipidating the creamy lipid with ethanol/ethel 3: 1. The proteins were dissolved with SDS-Tris buffer to 1mg protein/ml. This material was underwent SDS-polyacrylamide gel electrophoresis and the protein bands were visualized by placing gels into 4 M sodium acetate. Bands of the gels containing apoprotein A-IV were cut from the gels and pooled. Apoprotein A-IV was eluted from the gels with SDS-Tris buffer. The eluate was concentrated by ultrafiltration to 500μg/ml.
The antisera against apoprotein A-IV was produced by immunizing rabbits. Rocket immunoelectrophoresis for quantitative determination of human serum apoprotein A-IV was performed by using 1% agarose gel plates containing 2% (v/v) antisera.
Serum apoprotein A-IV levels of normal subjects (n=16) were 128±16u/dl. Apoprotein A-IV levels of 30 cases with diabetes mellitus were decreased to 109±30u/dl. The levels of apoprotein A-IV did not correlate either with serum lipids levels or clinical parameters such as fasting blood sugar levels, concentration of glycohemoglobin or grades of diabetic retinopathy. A case with short bowel syndrome showed a marked decreased level of serum apoprotein A-IV.
