Abstract
The degree of oxidized cysteine (Cys) 34 in human serum albumin (HSA), as determined by high performance liquid chromatography (HPLC), is correlated with oxidative stress related pathological conditions. In order to further characterize the oxidation of Cys34-HSA at the molecular level and to develop a suitable analytical method for a rapid and sensitive clinical laboratory analysis, the use of electrospray ionization time-of-flight mass spectrometer (ESI-TOFMS) was evaluated. A marked increase in the cysteinylation of Cys34 occurs in chronic liver and kidney diseases and diabetes mellitus. A significant positive correlation was observed between the Cys-Cys34-HSA fraction of plasma samples obtained from 229 patients, as determined by ESI-TOFMS, and the degree of oxidized Cys34-HSA determined by HPLC. The Cys-Cys34-HSA fraction was significantly increased with the progression of liver cirrhosis, and was reduced by branched chain amino acids (BCAA) treatment. The changes in the Cys-Cys34-HSA fraction were significantly correlated with the alternations of the plasma levels of advanced oxidized protein products, an oxidative stress marker for proteins. The binding ability of endogenous substances (bilirubin and tryptophan) and drugs (warfarin and diazepam) to HSA purified from chronic liver disease patients were significantly suppressed but significantly improved by BCAA supplementation. Interestingly, the changes in this physiological function of HSA in chronic liver disease were correlated with the Cys-Cys34-HSA fraction. In conclusion, ESI-TOFMS is a suitable high throughput method for the rapid and sensitive quantification of Cys-Cys34-HSA in a large number of samples for evaluating oxidative stress related chronic disease progression or in response to a treatment.
Highlights
Post-translationally modified proteins can be used as biomarkers for the diagnosis of diseases or for the assessment of treatment responses
Two major peaks, corresponding to Cys-Cys34-human serum albumin (HSA) and reduced Cys34-HSA, so-called HMA, were identified when plasma samples obtained from healthy subjects were analyzed by ESI-TOFMS (Figure 2A)
In agreement with previous findings, an increased Cys-Cys34-HSA peak accompanied by a decreased HMA peak was observed in patients with diabetes mellitus or chronic renal failure [19,27]
Summary
Post-translationally modified proteins can be used as biomarkers for the diagnosis of diseases or for the assessment of treatment responses. There is growing interest in developing diagnostic tools to monitor the extent of oxidative damage in tissues or organs, and the use of novel anti-oxidants for the treatment or prevention of such oxidative stress-related diseases [3,4,5]. At present, a rapid and sensitive clinical laboratory testing method for the rapid assessment of oxidative stress in humans is lacking. Since the redox status of thiol (SH) groups sensitively reflects the oxidation-reduction status of its surrounding environment, this HPLC analytical method has been shown to be useful for assessing the level of oxidative stress in diseased states and for evaluating the anti-oxidative activity of a therapeutic agent [8,9,10,11]
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