Cys34-cysteinylated human serum albumin is a sensitive plasma marker in oxidative stress-related chronic diseases

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

カンコウヘン カンジャ ニ オイテ ブンキ サ アミノサン セイザイ ケイコウ トウヨ ガ ヒト ケッセイ アルブミン ノ コウゾウ オヨビ キノウ カイゼン ニ オヨボス エイキョウ

熊本大

カンコウヘン カンジャ ニ オイテ ブンキ サ アミノサン セイザイ ケイコウ トウヨ ガ ヒト ケッセイ アルブミン ノ コウゾウ オヨビ キノウ カイゼン ニ オヨボス エイキョウ

博士(医学)熊本大

Deconvoluted ESI-TOFMS spectra of HSA.

<p>(A) Spectrum of HSA from healthy subject. (B) Spectrum of HSA from patient with chronic liver disease. (C) Spectrum of HSA from the same patient that (B) after DTT treatment. The peaks correspond to the following: (a) Asp-Ala truncation from N-terminal of HSA, (b) Leu truncation from C-terminal of HSA, (c) reduced HSA, (d) Cys-Cys34-HSA, (e) glycated HSA and (f) glycated Cys-Cys34-HSA.</p

Relationship between the Cys-Cys34-HSA fraction and the ligand unbound fraction in chronic liver disease patients.

<p>Effect of disease progression on the unbound fraction of warfarin (A), bilirubin (B), diazepam (C) and L-tryptophan (D) to purified HSA. Effect of BCAA treatment on the unbound fraction of warfarin (E), bilirubin (F), diazepam (G) and L-tryptophan (H) to purified HSA. Correlation between the Cys-Cys34-HSA fraction and the unbound fraction of warfarin (I), bilirubin (J), diazepam (K) and L-tryptophan (L) to purified HSA in chronic liver disease. Values are expressed as mean ± SD. *P<0.01 as compared with healthy subjects, ^#P<0.05 as compared with before treatment.</p

Relationship between the Cys-Cys34-HSA fraction (ESI-TOFMS) and the degree of oxidized Cys34-HSA (HPLC).

<p>The measured samples were 229 patients with chronic disease (including 139 patients with liver disease (circle), 38 patients with kidney disease (square) and 52 patients with diabetes mellitus (triangle)) (R² = 0.9025, P<0.001).</p

General characteristics of the study participants.

<p>Mean ± SD value are shown. BUN; blood urea nitrogen, SCr; serum creatinine, HD; hemodialysis.</p

The structure of HSA. Superposition of the Cys34 and the location of two major ligand binding sites, Site I and II are shown.

<p>The structure of HSA. Superposition of the Cys34 and the location of two major ligand binding sites, Site I and II are shown.</p

Change in the Cys-Cys34-HSA fraction and oxidative stress marker, AOPP, by BCAA treatment.

<p>(A) Change in the Cys-Cys34-HSA fraction. The Cys-Cys34-HSA fraction was measured by ESI-TOFMS. (B) Change in plasma AOPP level. (C) Correlation between the Cys-Cys34-HSA fraction and plasma AOPP level in chronic liver disease (n = 69). Values are expressed as mean ± SD (n = 20). *P<0.01 as compared with healthy subjects, ^#P<0.05 as compared with before treatment.</p

Effect of disease progression on the Cys-Cys34-HSA fraction in chronic liver disease patients.

<p>The Cys-Cys34-HSA fraction was measured by ESI-TOFMS. Values are expressed as mean ± SD (n = 9–79). *P<0.05 as compared with healthy subjects, ^#P<0.05 as compared with Child-Pugh Grade A, and ^§P<0.05 as compared with Child-Pugh Grade B.</p