A study was made of the interaction of plasma ascorbate and ascorbate free radical (AFR) with exogenously added iron. The quantitative determination of AFR has the advantage that transient increases in ascorbate oxidation can be directly monitored by e.p.r. spectroscopy. An AFR signal was found in the plasma of all donors and was unaffected by superoxide dismutase, catalase and the strong iron chelator deferoxamine. These findings and the rapid decrease in AFR under a nitrogen atmosphere suggest that plasma AFR is probably a result of air auto-oxidation. Iron loading of plasma did not affect the intensity of the AFR signal until the iron concentration approached or exceeded the plasma latent iron-binding capacity. In iron-overloaded plasma, the intensity of the AFR signal increased to about 10 times the normal level before decreasing rapidly to undetectable levels after 15-20 min. Determination of plasma ascorbate showed that the disappearance of AFR was due to a complete loss of the vitamin. When 50 microM-ascorbate was loaded with iron in iso-osmotic phosphate buffer there was an increase in the AFR signal, independent of the iron concentration, which was stable at least for 15 min. Thus the rate of ascorbate loss in the iso-osmotic phosphate buffer was considerably lower than in iron-overloaded plasma. The addition of different iron chelators produced comparable effects on the intensity of the AFR signal in both iron-overloaded plasma and ascorbate solution. These results suggest that the characteristic behaviour of plasma AFR after iron loading is due to its specific iron-binding capacity and to plasma ferroxidase activity. The ferroxidase activity of plasma is important to promote the transfer of Fe2+ into transferrin without a transient ascorbate oxidation. Spin-trapping studies with 5,5-dimethyl-1-pyrroline N-oxide and N-t-butyl-alpha-phenylnitrone revealed that iron-overloaded plasma was unable to produce spin-trap adducts even in the presence of 50-300 microM-hydrogen peroxide or 100 microM-azide. Evidence of OH. radical formation was obtained only after the addition of EDTA. Therefore, iron-overloaded plasma itself does not produce a Fenton reaction and, if ascorbate does indeed have a free-radical-mediated pro-oxidant role, it is not detectable in plasma by spin-trapping experiments.
Cultured rat hepatocytes were used to measure hepatic synthesis of rat plasma glycoproteins. [3H]Glucosamine was progressively incorporated into the protein of hepatocyte culture media very-low-density lipoprotein, low-density lipoprotein, high-density lipoprotein and the p greater than 1.21 g/ml fraction after 3.5 and 6.5 h incubation. Apolipoproteins B, E and C, as well as transferrin, were identified as glycoproteins. The association of radioactivity with apolipoprotein C of hepatocyte very-low-density and high-density lipoproteins suggests that apolipoprotein C-III-3, the only C apoglycoprotein in the rat, is synthesized de novo by the hepatocytes. Treatment of hepatocytes with tunicamycin, a specific inhibitor of protein glycosylation, resulted in a substantial decrease in [3H]glucosamine incorporation into hepatocyte very-low-density, low-density and high-density lipoproteins and p greater than 1.21 g/ml protein, but had little or no effect on secretion. In the rat, hepatic secretion of lipoproteins and transferrin does not appear to be dependent on prior protein glycosylation.