Abstract
Oxidative stress is a risk for and cause of various disease, however, measurements of oxidative stress are either time-consuming or non-specific. Here, we established a rapid method of using high performance liquid chromatography (HPLC) to measure serum oxidized albumin in a rat model. We optimized HPLC conditions for rat oxidized albumin. To validate our method, three-week-old male Sprague-Dawley rats were uninephrectomized and treated normal diet, high salt diet or high salt diet with Tempol, a superoxide dismutase (SOD) mimetic. After 4 weeks of treatment, we analyzed serum oxidized albumin. The main findings are listed as below. (i) Our method of oxidized albumin measurement only takes 16 minutes, with an intra-day and inter-day deviation within 1% and a detection limit concentration of 6.4 mg/ml. (ii) Oxidized albumin levels were significantly higher in the high salt diet group than in the normal salt diet group, and this effect was reversed by Tempol. (iii) Oxidized albumin levels also correlated with urinary protein and 8-isoprostane levels. In conclusion, we have established a simple method for evaluating rat serum oxidized albumin using HPLC. Our method is rapid and has an advantage over conventional methods and may be useful for animal models of oxidative stress.
Highlights
Oxidative stress has elicited high levels of interest in the field of biology for a long time
By systematically screening of measurement conditions, we eventually determined the optimal condition for measuring rat oxidized and reduced albumin: 25 mM phosphoric acid buffer with 60 mM sodium sulfate, plus 1.5% ethanol and 1000 mM magnesium chloride
We described a simple, adapted method for the measurement of rat oxidized albumin
Summary
Oxidative stress has elicited high levels of interest in the field of biology for a long time. Each of them has distinct characteristics, but does not necessarily reflect a ubiquitous oxidative stress level. A former study reported that oxidized rat serum albumin cannot be clearly separated from reduced albumin by conventional HPLC method[8]. Hayashi T et al has developed a method by HPLC which can separate both reduced and oxidize albumin in rat serum[9]. We established a simple and rapid method for measuring oxidized albumin in rat serum. We validated this method by using an established rat model of high oxidative stress which demonstrated proteinuria and hypertension[10,11,12]
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