Effect of High Glucose on the Production of Reactive Oxygen Species in Trabecular Meshwork Cells

PURPOSE: To investigate the effect of high glucose concentration on the production of reactive oxygen species in cultured human trabecular meshwork cells (HTMCs). METHODS: Primarily cultured HTMCs were exposed to low glucose (5 mM) and high glucose (22 mM) concentrations, respectively, for seven days. Cellular survival, as well as nitric oxide (NO) and hydrogen peroxide production, were assessed by measured MTT assay, Griess assay, and o-Dianisidine dihydrochloride assay, respectively. Some cells were co-exposed to N-acetyl cysteine (NAC) to assess the effect of this antioxidant. RESULTS: High glucose concentration increased the survival of cultured HTMCs significantly, with no effect from NAC. High glucose concentration increased the production of NO and hydrogen peroxide, which were abolished by co-exposure with NAC. CONCLUSIONS: High glucose concentration increases the production of NO and hydrogen peroxide, which can be abolished by antioxidant in trabecular meshwork cells.

Effect of mercuric chloride on kinetic properties of horseradish peroxidase

Mercury is one of the three major environmental metal poisons, and mercuric chloride is a highly reactive compound which can harm cells by a variety of mechanisms including direct interaction with sulphydryl groups of proteins and enzymes, therefore affecting the enzymatic activity. This study focused on the effect of Hg++ on horseradish peroxidase [donor: hydrogen peroxide oxidoreductase, EC 1.11.1.7] [HRP] [Isoenzyme C] activity. In the presence of 88 mM hydrogen peroxide Km for o-dianisidine oxidation was 0.05 millimolar and Vmax M.s-1. Incubation of the enzyme with 1 to 100 millimolar mercuric was 8.5 chloride for 5-20- and 60 min resulted in progressive inhibition of the enzymatic activity. At low Hg++ concentrations the inhibition was reversible by excess substrate, while at high Hg++ concentration the inhibition was not reversible. Results also indicated that the type of inhibition depended on the duration of incubation of the enzyme with metal ion and on the Hg++ concentration. So we could conclude that the type of inhibition changed from noncompetitive to mix with increased incubation time and increased metal concentration

Myeloperoxidase Assay on Hypoxic-Ischemic Brain Injury in Immature Rats / 대한소아신경학회지

PURPOSE: Neutrophils found around an infarcted area in the brain was once considered as only the physiologic response following the brain injury, but recent studies have shown that inflammatory responses by neutrophils play an important role in the reperfusion injury. The presence of polymorphonuclear leukocytes(PML) is proven by biochemical assay of myeloperoxidase(MPO) secreted in the cytoplasmic granules. We observed the process of PML infiltration on hypoxic-ischemic brain injury of immature rats by the assay of MPO activity and changes of the MPO activity after the administration of fucoidin, inhibitor of P- and L-selectin. METHODS: We used a well characterized model of the brains of 7 day-old-rats, which had unilateral hypoxic and ischemic injuries(HI). Those injuries were induced by unilateral carotid artery ligation followed by timed exposure to hypoxic inspiratory gas mixture(8% O2). MPO activity was measured in the brain tissue homogenates of HI rats(n=18) at 0, 2, 8, 24 and 48 hrs and in rats that received fucoidin immediately before and again after hypoxia(50 mg/kg, n=6) at 8 and 24 hrs. Controls(n=2) were rats with neither hypoxia nor ischemia. The brain samples were homogenized in 20 mM potassium phosphate buffer(pH 7.4) for 50 secs. The homogenate was centrifuged at 14,000 g at 4degrees C for 15 mins and the supernatant was discarded. The tissue was pulverized, weighed, and suspended in 1 mL of 50 mM potassium phosphate buffer solution(pH 6.0) containing 0.5% cetylditrimethylammonium bromide(wt/vol). The tissue was sonicated and centrifuged at 10,000 g for 15 mins. 200 micro L of the supernatant was mixed with 1 mL of 50 mM potassium phosphate buffer solution(pH 6.0) containing 10 micro L of 1.325 mM o-dianisidine hydrochloride and 170 micro L of 3% hydrogen peroxide(vol/vol). Changes in absorbance at 460 nm were measured for 5 mins by using microplate reader. One unit of MPO activity was defined as that degrading 1 micro mol peroxide/min at 25degrees C, and the result was expressed as units of MPO/100 mg tissue. RESULTS: In HI rats, MPO activity increased at 2 hrs after HI and peaked at 24 hrs in the right hemisphere. In rats with fucoidin treatment immediately before and again after hypoxia, the MPO activity significantly decreased in both hemispheres compared with HI rats(P<0.05). MPO activity in the tissue of control rats was insignificant. CONCLUSION: The dynamic changes of the MPO activity suggest the important role of PMN on hypoxic-ischemic brain injuries in immature rats. MPO activity could be used as an index of the severity of injuries of hypoxic-ischemic brains.