Serum protein-bound 3,4-dihydroxyphenylalanine and related products of protein oxidation and chronic hemodialysis.
BACKGROUND
Protein-bound dihydroxyphenylalanine (PB-DOPA) and its oxidation products are formed by free radical and oxidative attack on proteins. Hemodialysis and uremic toxins can activate leukocytes leading to overproduction of reactive oxygen species such as hydrogen peroxide and hypochlorous acid (HOCl) that increases protein oxidation.


METHODS
We have used a sensitive fluorometric method to measure PB-DOPA and its oxidation products in proteins after gamma-irradiation and incubation with HOCl and in serum from hemodialysis patients and healthy controls. These PB-DOPA concentrations were compared with those measured by HPLC (PB-DOPAHPLC).


RESULTS
Fluorescent PB-DOPA increased linearly with increasing amounts of human serum and with increasing amounts of gamma-irradiated bovine serum albumin. Concentrations of fluorescent PB-DOPA paralleled PB-DOPAHPLC levels but were approximately 60-70 times higher. Incubation of BSA and human serum albumin (HSA) with HOCl (39.4 mM) significantly (P < 0.0001) increased fluorescent PB-DOPA by 5 fold and 10 fold respectively and PB-DOPAHPLC by 6-fold for both proteins Fluorescent PB-DOPA concentration increased significantly (P < 0.0001) by 16-fold in human serum incubated with HOCl (39.4 mM). Mean serum fluorescent PB-DOPA was significantly (P < 0.0001) higher in 19 hemodialysis patients (57.7 +/- 16.1 microM) compared with 21 healthy controls (33.5 +/- 3.7 microM). Mean PB-DOPAHPLC was 4.45 +/- 1.63 microM in the healthy controls and 12 hemodialysis patients had values within the range of values in these controls while five patients had values that were outside eight SDs of the mean for healthy subjects. Serum fluorescent PB-DOPA was not correlated significantly with PB-DOPAHPLC in these subjects.


CONCLUSIONS
The results of this study suggest that fluorophores of the type, which are derived from DOPA can be reproducibly measured in delipidated serum protein and that HOCl can increase levels of these fluorophores-generating proteins and may potentially contribute to the high levels in serum from hemodialysis patients. This high level of fluorescent PB-DOPA compounds is only partially due to authentic PB-DOPA and might also be derived from other related protein oxidation products including those from DOPA oxidation.
Sutherland
Wayne H F
WH
Department of Medical and Surgical Sciences, University of Otago, Dunedin, New Zealand. wayne.Sutherland@stonebow.otago.ac.nz
Gieseg
Steven P
SP
Walker
Robert J
RJ
de Jong
Sylvia A
SA
Firth
Carole A
CA
Scott
Nicola
N
D000328:Adult; D000368:Aged; D015415:Biomarkers / Q000097:blood; D016022:Case-Control Studies; D002851:Chromatography, High Pressure Liquid; D004295:Dihydroxyphenylalanine / Q000032:analysis / Q000378:metabolism*; D005260:Female; D005470:Fluorometry; D005609:Free Radicals / Q000032:analysis; D006801:Humans; D007676:Kidney Failure, Chronic / Q000175:diagnosis / Q000628:therapy*; D008297:Male; D008875:Middle Aged; D018384:Oxidative Stress; D011336:Probability; D011379:Prognosis; D011485:Protein Binding / Q000502:physiology*; D012016:Reference Values; D006435:Renal Dialysis / Q000009:adverse effects* / Q000379:methods*; D018570:Risk Assessment; D012494:Sampling Studies; D018709:Statistics, Nonparametric
D015415:Biomarkers; D005609:Free Radicals; D004295:Dihydroxyphenylalanine