Clin Exp Nephrol (2009) 13:308315 DOI 10.

1007/s10157-009-0161-y

ORIGINAL ARTICLE

Albumin thiol oxidation and serum protein carbonyl formation are progressively enhanced with advancing stages of chronic kidney disease
Yukie Matsuyama Hiroyuki Terawaki Tomoyoshi Terada Seiichi Era

Received: 13 November 2008 / Accepted: 19 January 2009 / Published online: 11 April 2009 Japanese Society of Nephrology 2009

Abstract Background Oxidative stress is enhanced in advanced chronic kidney disease (CKD) patients and recognized as a main contributor to cardiovascular disease. Carbonyl stress is also known to be enhanced in advanced CKD; however the precise relationship between oxidative stress and carbonyl stress is not clear. The aim of this study was to investigate potential relationships between oxidative stress, carbonyl stress, and renal function among predialysis patients with CKD. Methods A total of 32 predialysis CKD patients (22 male, 10 female) were divided into four groups according to their values for creatinine clearance (Ccr) (group A, C60 ml/min; group B, 4559 ml/min; group C, 3044 ml/min; group D, B29 ml/min). As main markers of oxidative and carbonyl stresses, the redox state of Cys-34 (free thiol group) of human serum albumin [HSA(Cys-34)-redox] and the carbonyl content of serum proteins were employed, respectively. Results The values for the fraction of both reversibly oxidized HSA [f(HNA-1)] and irreversibly oxidized HSA [f(HNA-2)] signicantly increased with a decrease in renal
Y. Matsuyama T. Terada S. Era (&) Department of Physiology and Biophysics, Gifu University Graduate School of Medicine, 1-1 Yanagido, Gifu 501-1194, Japan e-mail: era@gifu-u.ac.jp H. Terawaki Research Division of Dialysis and Chronic Kidney Disease, Tohoku University Graduate School of Medicine, Sendai, Japan Present Address: H. Terawaki Division of Kidney and Hypertension, The Jikei University School of Medicine, Tokyo, Japan

function (group A, 21.0 3.4 and 1.8 0.3%; group D, 31.1 4.1 and 2.7 0.9%, respectively). The value for carbonyl content also signicantly increased with a decrease in renal function (group A, 0.7 0.1 nmol/mg protein; group D, 1.1 0.2 nmol/mg protein). There was a signicant positive correlation between carbonyl content and the f(HNA-2) value, while such a correlation was not observed between carbonyl content and the f(HNA-1) value, suggesting that there is a close relationship between serum protein carbonylation and irreversible albumin thiol oxidation. Conclusions There is a close relationship between oxidative stress and carbonyl stress and these are enhanced in correlation with the level of renal dysfunction among predialysis CKD patients. Keywords Albumin redox state Carbonyl formation Chronic kidney disease Oxidative stress Renal function

Introduction Cardiovascular disease (CVD) remains the most severe complication in patients with chronic kidney disease (CKD). Recent reports have demonstrated that large numbers of CKD patients become the victim of CVD before they reach dialysis, not only in Western populations [1] but also in Eastern populations [2, 3]. Therefore, the search to reveal the pathophysiology of CVD in CKD patients is an emerging worldwide problem. Oxidative stress is widely recognized as an important contributor to CVD among CKD patients. Until now, some investigators have reported that oxidative stress was enhanced among CKD patients in correlation with renal dysfunction [47]. Furthermore, other studies have

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reported that, while administration of antioxidant substances such as vitamin E and acetylcysteine reduced CVD in hemodialysis patients [8, 9], vitamin E did not exhibit effects in patients with no renal impairment [10]. Recently, Nanayakkara et al. [11] reported that an 18-month antioxidant treatment strategy consisting of pravastatin, vitamin E, and homocysteine-lowering therapy reduced carotid intima-media thickness (a strong surrogate marker of CVD risk) and urinary albumin excretion, and increased brachial artery ow-mediated dilatation (a marker of endothelial function). From these ndings, oxidative stress is, at least in part, involved in CVD among CKD patients, and this evidence seems very strong. In addition to oxidative stress, the elevation of carbonyl formation due to the long-term oxidative damage of lipids, carbohydrates, and proteins is also reported in several diseases, including CKD [12, 13]. Protein carbonylation is thought to reect carbonyl modication of amino acid residues, such as lysine, arginine, proline, and threonine, and it is the most general and commonly used biomarker of the state of long-term carbonyl overload or carbonyl stress. Miyata et al. [14] have noted that carbonyl formation in lipids, carbohydrates, and proteins could either be the result of oxidative reactions or of other nonenzymatic biochemical pathways in the long-term accumulation of uremic complications. However, the precise relationship between oxidative stress and protein carbonylation is as yet not clear. The aim of this study was to evaluate the relationship between oxidative stress and protein carbonylation in nondialysis CKD patients. In this study, we adopted the redox state of human serum albumin (HSA) as a marker of oxidative stress, and carbonyl content as a marker of protein carbonylation in serum. HSA is the most abundant extracellular protein, participating in a multitude of functions including transport of various molecules, maintenance of colloidal osmotic pressure, and buffering serum redox state [15]. The buffering function of HSA on serum redox state is due to a free cysteine residue at position 34 from the N-terminus (Cys34). The Cys-34 of HSA deoxidizes other substances according to the degree of surrounding oxidative stress and is itself oxidized (a sacricial antioxidant). From the perspective of this single cysteine residue, HSA is a mixture of human mercaptalbumin (HMA) in which the Cys-34 is not oxidized, human nonmercaptalbumin (HNA)-1, which is thought to have a disulde bond that can be reversibly oxidized by cysteine, and HNA-2, which is strongly oxidized and becomes a sulfenic (-SOH), sulnic (-SO2H), or sulfonic (-SO3H) species [5]. The Cys-34 of HSA makes up approximately 80% of the total free thiol content in plasma [15] and the state of Cys-34, i.e., the redox state of HSA, is thus indicative of the degree of short-term systemic oxidative stress because of the short half-life (*20 days) of

the albumin molecule [15]. We have developed a convenient high-performance liquid chromatography (HPLC) system for the clear separation of HSA into HMA and HNA [16, 17], and we have extensively studied the dynamic changes of HSA(Cys-34)-redox state under various physiologic [1820] and pathophysiologic states, such as hepatic [21, 22], renal [5, 2326], diabetic [27], and other diseases [2830]. We thus performed direct comparisons between each redox state of HSA(Cys-34) and the carbonyl formation of serum proteins in 32 nondialysis CKD patients with different levels of renal dysfunction within the CKD stage classication (stages 15). As a result, our study revealed a tight correlation between serum protein carbonyl formation, irreversible oxidation of Cys-34 of HSA (HNA-2 formation), and degree of renal dysfunction.

Materials and methods Patient characteristics The study involved 32 patients, 22 male and 10 female, aged between 32 and 92 years (60.9 15.2 years) with CKD. The local ethics committee approved this study protocol, and informed consent was obtained from all patients. CKD stage (stages 15) was classied by estimating the value for creatinine clearance (Ccr) using the CockcroftGault formula [31], and then patients were divided into the following four groups on the basis of estimated Ccr level: group A, C60 ml/min (CKD stages 1 and 2, n = 7); group B, 4559 ml/min (CKD stage 3a, n = 7); group C, 3044 ml/min (CKD stage 3b, n = 6); group D, B29 ml/min (CKD stages 4 and 5, n = 12) [1, 32], as shown in Fig. 1. Patient characteristics and primary kidney diseases are listed in Table 1. Physical measurements including height and
150

Ccr (ml/min)

100

50

stage 1 (n = 6)

stage 2 (n = 1)

stage 3a (n = 7)

stage 3b (n = 6)

stage 4 (n = 8)

stage 5 (n = 4)

group A ( n = 7)

group B

group C

group D ( n = 12)

Fig. 1 Mean creatinine clearance of each patient group. In CKD, the stage is classied by estimating the value for creatinine clearance using the CockcroftGault formula [31] (stages 1 to 5). Each column represents the mean value and each bar indicates standard deviation

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310 Table 1 Patient characteristics and clinical data group A (n = 7) Age (years) Gender (male/female) Height (cm) Weight (kg) Serum albumin (g/dl) Blood urea nitrogen (mg/dl) Serum creatinine (mg/dl) Creatinine clearance (ml/min) Primary disease CGN Not biopsy-proven MN FGS IgA nephropathy MPGN Diabetic nephropathy Nephrosclerosis Donor of RTx Lupus nephritis 2 2 1 0 1 0 0 0 1 1 0 1 2 1 0 2 0 0 1 0 2 1 0 1 0 1 0 51.3 7.6 5/2 165.7 9.9 68.9 8.8 4.0 0.1 16.4 3.4 0.7 0.1 117.6 30.7 group B (n = 7) 58.2 12.2 4/3 163.9 6.5 55.6 4.7 4.0 0.1 20.6 4.8 1.1 0.2 53.5 4.9

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group C (n = 6) 71.3 8.2 5/1 159.2 4.7 59.5 8.0 4.1 0.3 23.8 6.2 1.4 0.2 39.8 3.6

group D (n = 12) 63.3 19.4 8/4 161.2 9.1 57.7 9.5 4.1 0.3 49.6 16.4 3.8 2.9 19.2 7.6

4 0 0 4 0 3 1 0 0

Values expressed as numbers and mean standard deviations (SD) CGN chronic glomerulonephritis, MN membranous nephropathy, FGS focal glomerular sclerosis, MPGN membranoproliferative glomerulonephritis, RTx renal transplantation

weight were taken, and then blood samples were collected to measure the HSA redox state and carbonyl content. Five milliliters were drawn from serum obtained by centrifugation at 3,000 rpm for 15 min at 4C and stored at -80C until analysis. In addition, the following biochemical tests were measured with standard laboratory techniques in a single laboratory: serum concentrations of albumin, blood urea nitrogen (BUN), and creatinine. None of the patients had received antioxidant agents such as ascorbic acid (AsA) or vitamin E. Patients with infection, inammatory reaction, bleeding, liver dysfunction or malignancies were not included in the present study. In all diabetic subjects, the value for HbA1c was lower than 6.5%. HPLC system for measurement of the albumin redox state Measurement of the albumin redox state was performed by the HPLC method previously reported [26]. Briey, the HPLC system consisted of an AS-8010 autosampler (injection volume of 2 ll of specimen) and a CCPM double-plunger pump in conjunction with an SC-8020 system controller (all from Tosoh, Tokyo, Japan). The chromatograph was obtained with a Finnigan UV6000LP photodiode array detector (detection range 200600 nm with 1-nm step, Thermo Electron, Waltham, MA, USA).

A Shodex-Asahipak ES-502N 7C column (10 9 0.76 cm I.D., DEAE-form for ion-exchange HPLC; Showa Denko, Tokyo, Japan; column temperature, 35 0.5C) was used in this study. Elution was performed by linear gradient elution with ethanol (at the following concentrations: 01 min, 0%; 150 min, 0% ? 10%; 5055 min, 10% ? 0%; 5560 min, 0%) for specimen in 0.05 M sodium acetate and 0.40 M sodium sulfate mixture (pH 4.85) at ow rate of 1.0 ml/min. Deaeration of the buffer solution was performed by bubbling helium through it. HPLC proles obtained from these procedures were subjected to numerical curve tting with simulation software (PeakFit, version 4.05, SPSS Science, Chicago, IL, USA); each peak shape was approximated by a Gaussian function (Fig. 2). The values for the fractions of HMA [f(HMA)], HNA-1 [f(HNA-1)], and HNA-2 [f(HNA-2)] were obtained by the following equations: f HMA% HMA=HMA HNA-1 HNA-2 100; f HNA-1% HNA-1=HMA HNA-1 HNA-2 100; f HNA-2 % HNA-2=HMA HNA-1 HNA-2 100 :

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a 100
HMA
intensity (mAU)

b
HMA: 77.9 % HNA-1: 21.3 % HNA-2: 0.8 %
intensity (mAU)

100

HMA: 66.7 % HNA-1: 29.9 % HNA-2: 3.4 %

HMA

50

50
HNA-1 HNA-2

HNA-1 HNA-2

0 0 10 time (min) 20 30

10
time (min)

20

30

Fig. 2 HPLC proles of serum from one of the patients in group A (a) and group D (b) eluted from an ES-502 N column with an ethanol concentration increasing from 0% to 10% in acetate-sulfate buffer (pH 4.85). Peaks 1, 2, and 3 correspond to HMA, HNA-1, and HNA-2,

respectively. The proles are subjected to numerical curve tting (dashed line), and the obtained values for each fraction are indicated in the upper right part of the gure

Assay system for carbonyl content measurement Carbonyl content was determined using the Protein Carbonyl Assay Kit according to the manufacturers instructions (Cayman Chemical Co., USA). This kit utilizes the 2,4-dinitrophenylhydrazine (DNPH) reaction to measure carbonyl content. The amount of protein-hydrozone produced is quantied spectrophotometrically at an absorbance of 370 nm (Smart Spec Plus spectrophotometer, BIORAD Laboratories, Inc., USA). Statistical analysis Values were expressed as mean standard deviation unless otherwise stated. We used the statistical software Stat View 5.0 (SAS Institute, Inc., Cary, NC, USA). The chi-squared test was used for categorical variables, whereas comparisons of means among the four groups were analyzed by using one-way analysis of variance (ANOVA). After ANOVA, TurkeyKramers post hoc test was used for comparisons between groups. To measure the magnitude of correlation, we used Pearsons correlation coefcient (R). The correlation was determined to be signicant when the P value was less than 0.05 (5%) with Fishers Z transformation.

100 80

*
f (HMA) (%)
60 40 20 0

group A (n = 7)

group B (n = 7)

group C (n = 6)

group D (n = 12)

*, P < 0.05 vs. group A

Fig. 3 Comparison of f(HMA) values (%) between groups (P \ 0.0001, one-factor ANOVA). When the disease stage was advanced, the mean value for f(HMA) decreased as CKD stage progressed (group A, 77.2 3.4; group B, 75.5 3.8; group C, 71.5 3.3; group D, 66.2 4.1%). There was a signicant difference between groups A and D (P \ 0.05, TurkeyKramers test). Each column represents the mean value and each bar indicates standard deviation

Results HSA(Cys-34)-redox Figure 2 shows typical HPLC serum proles from one of the patients in groups A (Fig. 2a) and D (Fig. 2b),

respectively. The obtained values for f(HMA), f(HNA-1), and f(HNA-2) were 77.9%, 21.3%, and 0.8% for the patient in group A, and 66.7%, 29.9%, and 3.4% for the patient in group D. These HSA(Cys-34)-redox state HPLC results are summarized in Figs. 3 and 4. As shown in Fig. 3, the mean value for f(HMA) decreased progressively with a decrease in renal function [group A (n = 7), 77.2 3.4; group B (n = 7), 75.5 3.8; group C (n = 6), 71.5 3.3; group D (n = 12), 66.2 4.1%]. There was a signicant difference between groups A and D (P \ 0.05, TurkeyKramers test). The

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a
f (HNA-1) (%)

50 40 30 20 10 0

b
f (HNA-2) (%)

5 4 3 2 1 0

group A (n = 7)

group B (n = 7)

group C (n = 6)

group D (n = 12)

group A (n = 7)

group B (n = 7)

group C (n = 6)

group D (n = 12)

*, P < 0.05 vs. group A

Fig. 4 Comparison of f(HNA-1) and f(HNA-2) values (%) between groups. The mean value of f(HNA-1) increased as CKD stage progressed (group A, 21.0 3.4; group B, 22.4 4.1; group C, 26.2 3.1; group D, 31.1 4.1%). There was a signicant difference between groups A and D (P \ 0.05, TurkeyKramers test) (a; P \ 0.0001, one-factor ANOVA). The mean value for f(HNA-2) also increased in a similar manner (group A, 1.8 0.3; group B, 2.1 0.6; group C, 2.3 0.4; group D, 2.7 0.9%). There was a signicant difference between groups A and D (P \ 0.05, Turkey Kramers test) (b; P = 0.01, one-factor ANOVA). Each column represents the mean value and each bar indicates standard deviation

mean value for f(HNA-1), on the contrary, increased progressively with a decrease of renal function (group A, 21.0 3.4; group B, 22.4 4.1; group C, 26.2 3.1; group D, 31.1 4.1%). There was a signicant difference between groups A and D (P \ 0.05, TurkeyKramers test) (Fig. 4a). Likewise, that for f(HNA-2) followed a similar trend (group A, 1.8 0.3; group B, 2.1 0.6; group C, 2.3 0.4; group D, 2.7 0.9%). There was a signicant difference between groups A and D (P \ 0.05, Turkey Kramers test) (Fig. 4b). Carbonyl content of serum proteins The carbonyl content of serum proteins also progressively increased with a decrease in renal function (group A, 0.7 0.1; group B, 0.8 0.1; group C, 1.0 0.2; group D, 1.1 0.2 nmol/mg protein). There was a signicant difference between groups A and D (P \ 0.05, TurkeyKramers test) (Fig. 5). Correlation between oxidized HSA, carbonyl content, and clinical data The relationships between the oxidized HSA (HNA-1 and HNA-2), carbonyl content, and clinical data, such as BUN, serum creatinine, and Ccr, are given in Table 2. A signicant positive correlation was observed between the value for BUN (mg/dl) and those for f(HNA-1) (R = 0.605, P = 0.0002), f(HNA-2) (R = 0.704, P \ 0.0001), and carbonyl content (R = 0.695, P \ 0.0001). A signicant positive correlation was also observed between the value for serum creatinine (mg/dl) and those for f(HNA-1) (R = 0.546,

carbonyl content (nmol/mg protein)

1.5

0.5

group A (n = 7)

group B (n = 7)

group C (n = 6)

group D (n = 12) *, P < 0.05 vs. group A

Fig. 5 Comparison of carbonyl content of serum proteins (nmol/mg protein) between groups (P \ 0.0001, one-factor ANOVA). Carbonyl content progressively increased with CKD stage (group A, 0.7 0.1; group B, 0.8 0.1; group C, 1.0 0.2; group D, 1.1 0.2 nmol/ mg protein). There was a signicant difference between groups A and D (P \ 0.05, TurkeyKramers test). Each column represents the mean value and each bar indicates standard deviation

P = 0.001), f(HNA-2) (R = 0.668, P \ 0.0001), and carbonyl content (R = 0.523, P = 0.0018). In contrast, a signicant negative correlation was observed between the value for Ccr (ml/min) and those for f(HNA-1) (R = -0.617, P = 0.0001), f(HNA-2) (R = -0.487, P = 0.0041), and carbonyl content (R = -0.692, P \ 0.0001). Direct correlation between oxidized HSA and carbonyl content in serum In the relationship between albumin thiol oxidation and carbonyl formation in serum, the value for carbonyl content was correlated signicantly not with that for f(HNA-1)

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(R = 0.317, P = 0.08) (Fig. 6a) but with that for f(HNA-2) (R = 0.626, P \ 0.0001) (Fig. 6b), as detected by linear regression analysis. Thus, a close relationship was observed between carbonyl content of serum proteins and irreversible albumin thiol oxidation among predialysis patients with CKD.

Discussion Recent reports revealed that oxidative stress in CKD patients is elevated in accordance with the degree of renal dysfunction. Dounousi et al. [7] reported enhanced oxidative stress in advanced CKD patients using the serum level of 8-isoprostanes (8-epiPGE2a), considered to be a marker of endogenous lipid peroxidation. We also previously reported that the Cys-34 of HSA is oxidized in correlation with renal dysfunction [5], and that dialyzable uremic solutes might contribute to such HSA oxidation in a clinical setting [25, 26]. A large amount of HSA is synthesized only in the liver every day and circulates throughout the body. As already mentioned, HSA forms a major part of the redox potential of the body, because a

Table 2 Correlation between f(HNA-1), f(HNA-2), carbonyl content, and clinical data f(HNA-1) R BUN Creatinine Ccr f(HNA-2) P value R 0.605* 0.0002 0.546* 0.001 -0.617* 0.0001 Carbonyl content P value R 0.704* \0.0001 0.668* \0.0001 -0.487* P value 0.695* \0.0001 0.523* 0.0018

0.0041 -0.692* \0.0001

* P \ 0.05; n = 32

large portion of the thiol groups in human extracellular uid is primarily derived from the Cys-34 in HSA. From the viewpoint of thiol-redox state, HSA is composed of HMA (reduced form) and HNA (oxidized form). Further, HNA consists of HNA-1 (a major fraction, reversibly oxidized form) and HNA-2 (a minor fraction, irreversibly oxidized form). The present study provides several lines of evidence to suggest that the redox state of HSA(Cys-34) progressively changes with renal dysfunction as indicated by the CKD stage classication. The mean value for f(HMA) decreased progressively with a decrease in renal function; conversely the values for both f(HNA-1) and f(HNA-2) increased progressively with a decrease in renal function. In the light of our ndings for oxidized albumin, a close correlation was observed between the value for f(HNA-1) or f(HNA-2) and uremic toxins, such as BUN and creatinine. To date, it is still unclear why a decrease in renal function promotes oxidative stress. Although there are many reports about the relationship of in vivo antioxidant enzymes and renal function, opinions seem to diverge when it comes to analyzing the relationship between renal function, antioxidant enzyme activity of the renal cortex, and that of the serum [3338]. Meanwhile, accumulated uremic toxins themselves may induce oxidative stress. It is reported that by performing dialysis on patients with renal failure, decreased serum glucose-6-phosphate dehydrogenase, superoxide dismutase, and catalase activity all began to show normal values [33], and the proportion of oxidized albumin began to decrease [16, 21]. Protein carbonylation is also reported to be elevated in advanced CKD, not only in end stage, but also in the predialysis stage. Hou et al. [39] reported that the plasma level of pentosidine (one of the representative advanced glycation end-products; AGE) and the expression of the

45 40

R = 0.317, P = 0.08

5 4

R = 0.626, P < 0.0001

(HNA-2) = 0.306+2.041 carbonyl content; R2 = 0.392

f ( HNA-1) (%)

35 30 25 20 15

f ( HNA-2) (%)
.4 .6 .8 1 1.2 1.4 1.6

3 2 1 0

.4

.6

.8

1.2

1.4

1.6

carbonyl content (nmol/mg protein)

carbonyl content (nmol/mg protein)

Fig. 6 Relationships between carbonyl content and values for f(HNA-1) or f(HNA-2) in 32 predialysis patients with CKD (group A, open triangles; group B, open circles; group C, closed triangles; group D, closed circles). a Carbonyl content versus f(HNA-1)

(R = 0.317, P = 0.08). b Carbonyl content versus f(HNA-2) (R = 0.626, P \ 0.0001). There is a strong and highly signicant relationship between carbonyl content and f(HNA-2)

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receptor for AGE (RAGE) both increased in accordance with the degree of renal dysfunction. Miyata et al. [14] mentioned in their review that proteins are progressively and irreversibly modied by AGE and almost all of these modied proteins are thought to be serum albumin. This process is markedly accelerated not only in diabetes but also in uremic complications. In the present study, we investigated the relationship between HSA(Cys-34)-redox state and serum protein carbonylation in predialysis CKD patients and we found a strong relationship between irreversible oxidation of HSA(Cys-34) and serum protein carbonylation in accordance with the degree of renal dysfunction. Although such a relationship in patients with liver diseases was recently reported as an abstract only by Stauber et al. [40], our present work is the rst report which reveals a close correlation between f(HNA-2), serum protein carbonylation, and degree of renal dysfunction in predialysis CKD patients. These results also suggest that, at least in part, the irreversible thiol oxidation of albumin observed in predialysis patients might be involved in carbonyl formation of serum proteins that occurs in the long-term accumulation of uremic complications. There are some limitations to this study. One limitation is the method for the estimation of the patients renal function. We did not employ the recently recommended isotope dilution mass spectrometry-derived new modication of diet in renal disease (MDRD) study equation [41] to estimate the glomerular ltration rate (GFR) because a reliable racial coefcient for calculating the MDRD equation for the Japanese population is not known as yet. Moreover, a recent report suggests that the MDRD equation might not be adapted for non-Caucasian populations [42]. Therefore, we estimated GFR using CockcroftGault equation [31] in this study. Another limitation, probably a more important one, is that the number of evaluated patients was relatively small (32 patients). A larger number of patients might, of course, be required to perform a more accurate statistical analysis regarding confounding factors, such as age, sex, and primary disease of CKD. At present, as CKD patients are known to develop CVD and the number of patients with CKD is gradually increasing, the search for early clinical treatments and methods for early detection of CKD are some of the most important areas in this eld. As albumin participates in the maintenance of redox potential in the extracellular uids, and as almost all carbonylated serum proteins are thought to be albumin, simultaneous analysis of HSA(Cys-34)redox state and carbonyl content of serum proteins used in this study might be one of the useful methods for the detection of renal dysfunction.

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