Sodium dodecyl sulphate gel electrophoretic preparation of protein standard human apolipoprotein B-48.

Quantitation of plasma apo B-48 is currently performed by densitometric analysis of SDS-PAGE zones stained with Coomassie Brilliant Blue, using standard solutions of purified apo B-48. Here, preparative gel electrophoresis with a continuous elution system was used for purifying apo B-48. A chylomicron fraction was isolated by 107,000 g ultracentrifugation of a chylous ascite. The proteins were delipidated and precipitated in ethanol-diethyl ether (3:1, v/v), subjected to preparative electrophoresis in a 5% polyacrylamide gel and eluted in 0.1% SDS. The peak containing apo B-48 was eluted at a retention time of 445-480 min. The purity of apo B-48 in this fraction was assessed by the detection of a single band (M(r) 260,000) after silver staining and Coomassie staining of 4-15% gradient SDS-PAGE. It was confirmed by the absence of apo B-100 contaminant in Western blot of the purified protein preparation. A linear relationship was observed between the densitometric analysis of SDS-PAGE bands and the apo B-48 in a protein range of 0-3 microg. In conclusion, preparative gel electrophoresis was used in a single step purification of apo B-48 that was adapted to the preparation of a standard solution.

Diagnosis of lipid malabsorption in patients with chronic pancreatitis: a new indirect test using postprandial plasma apolipoprotein B-48.

OBJECTIVE: This study was done to evaluate a new test for fat malabsorption caused by chronic pancreatitis. Postprandial plasma apolipoprotein B-48 was measured as an indicator of the intestinal assimilation of exogenous lipid. METHODS: Twenty-three patients with chronic pancreatitis, including 19 insulin-treated diabetic patients, were compared with 14 healthy subjects and seven type-1 diabetic patients. Each was given a test meal containing 40 g lipid; the triglyceride apolipoprotein B-48 concentrations in chylomicrons were determined 240 min later. RESULTS: The postprandial chylomicron apolipoprotein B-48 concentrations in the three groups were statistically different at 240 min: pancreatitis versus controls, p < 0.01, and pancreatitis versus type-1 diabetes subjects, p < 0.01. The delta plasma apo B-48, the change in apolipoprotein B-48 between 0 and 240 min, was significantly smaller in chronic pancreatitis patients than in controls (p < 0.001) and type-1 diabetes subjects (p < 0.001). The sensitivity of the test was better than 89% for a delta apo B-48 threshold value of 0.42 mg/dl. CONCLUSIONS: This new indirect test is relatively simple to use and could be practical for evaluating exogenous lipid malabsorption due to chronic pancreatitis.