ISOLATION OF ACUTE-PHASE PROTEINS FROM PLASMA FOR DETERMINATION OF FRACTIONAL SYNTHESIS RATES BY A STABLE-ISOTOPE TRACER TECHNIQUE

Because of the unavailability of convenient tracer methods, there is n o information on the kinetic changes responsible for the increased or decreased pool sizes of the acute-phase proteins (APPs) during the met abolic response to trauma and infections. We have developed a stable i sotope tracer method to measure the synthesis rates of the APPs transf errin, haptoglobin, and alpha 1-antitrypsin, The proteins were isolate d from plasma by either direct or sequential immunoprecipitation and p urified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With the exception of haptoglobin, the major band of each protein and its standard ran to a position corresponding to its molecular mass. Th e major band of haptoglobin and its standard ran to a position corresp onding to a molecular mass of slightly less than 44 kDa, which corresp onds to the haptoglobin beta chain, molecular mass 42.5 kDa. To measur e the fractional synthesis rates (FSRs) of these proteins, three child ren were infused with H-2(3)-leucine for 8 h, and the amount of H-2(3) -leucine incorporated into the proteins was measured by gas chromatogr aphy-mass spectrometry. The plateau isotopic enrichment of very low de nsity lipoprotein-apoB-100-bound leucine was used to estimate the isot opic enrichment of the hepatic protein synthetic precursor pool. FSRs (%/day) of the proteins were haptoglobin, 50 +/- 8; transferrin, 19 +/ - 1.1; and alpha 1-antitrypsin, 10 +/- 1.5. (C) 1996 Academic Press, I nc.