Differences in the accumulation of ascorbic acid in normal, myeloperoxidase deficient and NADPH-oxidase deficient granulocytes.

Granulocytes contain large quantities of ascorbic acid (AA). The uptake mechanism is mainly restricted to the accumulation of the oxidized form, dehydroascorbate (DHA). We investigated the uptake of ascorbic acid and dehydroascorbate of normal, myeloperoxidase (MPO)-deficient, and NADPH-oxidase-deficient granulocytes. The accumulation of ascorbic acid was increased in all types of granulocytes after stimulation with phorbol-myristate-acetate, whereas the NADPH-oxidase-deficient cells showed a decreased uptake compared to normal and MPO-deficient cells. The intracellular concentration of ascorbic acid was further enhanced after incubation of granulocytes with DHA, most prominently in NADPH-oxidase-deficient granulocytes. MPO-deficient granulocytes are not able to produce HOCl after activation. The granulocytes of one individual with total MPO deficiency accumulated ascorbate in higher concentrations than did cells with partial MPO deficiency, indicating that HOCl is of minor importance for the oxidation of ascorbate. Since the ability of MPO-deficient cells to kill microorganisms is pronounced in contrast to NADPH-oxidase-deficient cells, effective mechanisms of compensating for the absence of HOCl must exist. We hypothesize that the enhanced uptake of ascorbic acid combined with an enhanced superoxide anion production may favor the generation of OH radicals via the Fenton reaction.

Low-density lipoprotein modification by normal, myeloperoxidase-deficient and NADPH oxidase-deficient granulocytes and the impact of redox active transition metal ions.

The modification of low-density lipoprotein (LDL) by normal, myeloperoxidase (MPO)-deficient and NADPH oxidase-deficient granulocytes was investigated using the monoclonal antibody (mAb) OB/04, which was originally generated against copper-oxidized LDL. Incubation of LDL with normal granulocytes increased the reactivity of LDL with mAb OB/04. These effects were even more pronounced using MPO-deficient granulocytes. Inhibitors of oxidative reactions (the NADPH oxidase inhibitor diphenyleneiodonium chloride [DPI], catalase, superoxide dismutase [SOD]) did not significantly reduce LDL oxidation by normal granulocytes. Furthermore, granulocytes of a patient with NADPH oxidase deficiency were almost equally effective as normal granulocytes, indicating that oxidative burst-derived reactive oxygen species are of only minor importance in the generation of mAb OB/04-detectable new epitopes on LDL in vitro. In contrast, incubation of LDL with iron and copper prior to and during incubation with normal granulocytes markedly enhanced the generation of OB/04-detectable epitopes. It is supposed that, besides superoxide (in normal and MPO-deficient granulocytes) or instead of superoxide (in NADPH oxidase-deficient granulocytes), lytic enzymes released by activated granulocytes may enhance the availability of transition metals for oxidation of LDL. Our results support the concept that transition-metal-dependent pathways of LDL oxidation in combination with degranulation products of granulocytes are important.