Hemolysis Of Human RBC Induced By High Concentration Of Ascorbic Acid In Vitro And Its Clinic Significance

Objective:To investigate the action and mechanism of ascorbic acid (Vitamin C) at high concentration on hemolysis of human red blood cell (RBC) in vitro by measuring the release of hemoglobin (Hb) , and the difference of sensitivity to this induced hemolysis of RBC from individuals of different disease states. Methods1. Hemolyzed rabbit RBC was used as the sample to evaluate the resistance of different methods for the measurement of Hb to the interferences of ascorbic acid and common inhibitors of intracellular antioxidation components. The interferences of common inhibitors of the antioxidation system and ascorbic acid on the assay of Hb by its absorbance at 540 nm, alkalinized Hb in complex to hydroxyl ion by absorbance at 575 nm, or the azide-Hb complex by absorbance at 542 nm were compared.2. By measuring hydroxylated Hb, the time course of induced hemolysis, the dependence of hemolysis on ascorbic acid concentration were investigated, in the presence and absence of inhibitors of the intracellular antioxidation system of human RBC to reveal the role of intracellular antioxidation system in ascorbate-induced hemolysis.3. The difference of spontaneous and ascorbic acid-induced hemolysis in RBC from individuals of different ages (the youth, the middle-aged andthe elderly), and some diseases states (Hypertension, Coronary Atherosclerotic Heart Disease and Diabetes mellitus) were investigated to reveal the clinic importance of hemolysis induced by ascorbic acid at the high concentration. Results1. Ascorbic acid and the inhibitors of superoxide dismutase, catalase exhibited significant interferences with direct assay of Hb by absorbance at 540 nm and the assay of azide-Hb, and blocker of GSH, inhibitor of GSH peroxidase showed smaller effects. The assay of alkalinized Hb exhibited the strongest resistance to all these chemicals.2. Both the spontaneous and induced by 2.0 mmol/L ascorbic acid alone hemolysis were lower than those in the presence of inhibitors of intracellular antioxidant enzymes in RBC.3. There was duration-dependent increase of hemolysis of human RBC in vitro in the presence of ascorbic acid at 2.0 mmol/L, in the presence or absence of inhibitors of intracellular antioxidation system, which becomes significant after 3.5 hr treatment. There was also much significant dependence of hemolysis on ascorbic acid concentrations in the presence of inhibitors on intracellular antioxidation system.4. The inhibitor alone on either superxoide dismutase (Diethyldithiocarbamate, DDC), glutathione peroxidase (Carmustine, CAR), catalase (Aminotrazole, ATA) or even glutathione itself (Iodoacetamide, IAA) significantly promoted ascorbic acid-induced hemolysis. But the combination of IAA, DDC, CAR and ATA to inhibit intracellular antioxidation system showed much stronger action to promote spontaneous hemolysis and induced hemolysis at 2.0 mmol/L Ascorbic acid. Among the combinations of two inhibitors, ATA and CAR were more effective to promote spontaneous hemolysis while DDC and ATA were more effective to promote ascorbicacid-induced hemolysis. And among the combinations of three inhibitors, IAA, DDC and CAR was most effective to promote spontaneous hemolysis induced by ascorbic acid at 2.0 mmol/Lo The combination of these four inhibitors showed the highest effects on spontaneous hemolysis and induced hemolysis at 2.0 mmol/L Ascorbic acid.5. Lipid peroxides on RBC membrane correlated well to the degree of RBC hemolysis indexed as the amount of Hb leaked out (P <0.01).6. High concentrations of ascorbic acid induced hemolysis to RBC from individuals, either the health or patient of different ages. There were increases of spontaneous and induced-hemolysis of RBC at 2.0 mmol/L Ascorbic acid from individual above middle-age. If antioxidant enzymes in RBC were inhibited, there was much stronger increase of spontaneous hemolysis of human RBC in the absence of ascorbic acid, and there was much higher sensitivity of RBC to high concentrations of ascorbic acid. RBC from patients of circulating system disease was more sensitive to Ascorbic acid-induced hemolysis.Conclusions1. The assay of alkalinized Hb was desirable for the investigation of the action and mechanism of peroxidative hemolysis induced by Ascorbic acid.2. It is the intracellular antioxidative system that determines the sensitivity of RBC to spontaneous hemolysis and induced hemolysis by high concentrations of ascorbic acid. The interaction between ascorbic acid and hemoglobin was possible to produce reactive oxygen species and promote hemolysis.3. There was decrease of intracellular antioxidation activites of human RBC with the increase of age, and concomitantly there was increase of the sensitivity of RBC to spontaneous and ascorbic acid-induced...