Hypochlorous acid and low serum paraoxonase activity in haemodialysis patients: an in vitro study.
BACKGROUND
Serum paraoxonase 1 (PON1) is an oxidant-sensitive enzyme associated with high-density lipoprotein (HDL) that inhibits the atherogenic oxidation of low-density lipoprotein (LDL). In haemodialysis patients, production of reactive oxygen species, such as hypochlorous acid (HOCl) and hydrogen peroxide, is increased and serum PON1 arylesterase is abnormally low. We have examined the effect of HOCl and the uraemic milieu on serum PON1 arylesterase activity and the ability of HDL to inhibit LDL oxidation in vitro.


METHODS
Serum was incubated with HOCl, hydrogen peroxide and products of HOCl reaction with excess cysteine, lysine and taurine and then serum PON1 arylesterase and serum protein tryptophan fluorescence were measured. The ability of plasma HDL fractions isolated by a dextran-sulphate method, to protect LDL from mild oxidation in air, was determined by a fluorimetric method using oxidation of 2,7-dichlorofluorescein (DCFH).


RESULTS
Incubation of healthy serum with HOCl in the range 6.5-32.9 mmol/l resulted in a linear decrease in serum PON1 arylesterase activity to 40% of that without HOCl and a parallel decrease in protein tryptophan fluorescence. The HOCl-induced decrease in serum PON1 activity was completely removed by reaction of HOCl with a 2.7-fold excess of alpha-amino acids but not taurine. In serum incubated for 1 week, the decrease in serum PON1 activity was significantly (P = 0.04) less while the increase in protein fluorescent advanced glycation end-products was significantly larger (P = 0.01) in haemodialysis patients compared with healthy subjects. The mean decrease in mild oxidation of LDL was not significantly different on addition of HDL-rich fractions from haemodialysis patients (100 +/- 6%, n = 7) and healthy subjects (95 +/- 6%, n = 7) or on addition of the HDL-rich fraction from plasma treated with 0.95 mmol/l HOCl (95%) and control HDL (96%). The fraction rich in HDL and other high molecular weight compounds from plasma that had been incubated with increasing HOCl concentrations up to 1.9 mmol/l significantly (P = 0.001) increased (471%) the oxidation of DCFH.


CONCLUSIONS
These results suggest that high concentrations of HOCl that severely oxidize serum proteins and tryptophan residues in the active site of PON1 are required to decrease PON1 arylesterase activity in serum. In haemodialysis patients, overproduction of HOCl that leads to high concentrations of severely oxidized proteins and increased oxidants in plasma might also contribute to low serum PON1 arylesterase activity, but does not appear to impair the ability of an HDL molecule to protect LDL from mild oxidation.
Sutherland
Wayne H F
WH
Department of Medical and Surgical Sciences, University of Otago Medical School, PO Box 913, Dunedin, New Zealand. wayne.sutherland@stonebow.otago.ac.nz
de Jong
Sylvia A
SA
Walker
Robert J
RJ
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D043303:Aryldialkylphosphatase / Q000378:metabolism*; D002784:Cholesterol / Q000378:metabolism*; D008076:Cholesterol, HDL / Q000187:drug effects / Q000378:metabolism; D008078:Cholesterol, LDL / Q000187:drug effects / Q000378:metabolism; D005260:Female; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D006997:Hypochlorous Acid / Q000494:pharmacology*; D066298:In Vitro Techniques; D007676:Kidney Failure, Chronic / Q000097:blood / Q000378:metabolism / Q000628:therapy; D008297:Male; D008875:Middle Aged; D016877:Oxidants / Q000494:pharmacology*; D018384:Oxidative Stress / Q000502:physiology; D006435:Renal Dialysis; D044967:Serum / Q000187:drug effects* / Q000378:metabolism
D008076:Cholesterol, HDL; D008078:Cholesterol, LDL; D016877:Oxidants; D006997:Hypochlorous Acid; D002784:Cholesterol; D006861:Hydrogen Peroxide; D043303:Aryldialkylphosphatase; C468406:PON1 protein, human