Key words

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Connection Between Ischemia−Modified Albumin Levels

and Markers of Diabetic Nephropathy and Oxidative

Protein Damage in Type 2 Diabetic Patients

Związek albuminy modyfikowanej niedokrwieniem

z markerami nefropatii cukrzycowej i oksydacyjnego uszkodzenia

białek u chorych na cukrzycę typu 2

1_{Department of Pharmaceutical Biochemistry, Wroclaw Medical University, Poland}

2_{Department and Clinic of Angiology, Hypertension and Diabetology, Wroclaw Medical University, Poland}

Adv Clin Exp Med 2009, 18, 4, 353–360 ISSN 1230−025X

ORIGINAL PAPERS

© Copyright by Wroclaw Medical University

Abstract

Background.Ischaemia and hypoxia are observed in diabetic patients in addition to chronic hyperglycemia and intensified oxidative stress. It is reflected by increased markers levels of oxidative protein damage, especially in patients with vascular complications, as well as ischemia−modified albumin (IMA).

Objectives. To assess the plasma levels of IMA in T2DM patients with different degrees of diabetic nephropathy and their relationship with routine markers of kidney disease severity (urinary albumin/creatinine, plasma creati− nine concentration) and parameters of oxidative processes, i.e. advanced oxidation protein products (AOPPs) and sufhydryl (SH) and carbonyl (CO) groups.

Material and Methods.Plasma IMA levels were manually determined in 78 patients and 22 healthy people by spectrophotometric Co(II)−albumin binding assay. Diabetic nephropathy stage was determined on the basis of the urinary albumin/creatinine ratio. Plasma AOPP levels as well as SH and CO groups (as DNPH reactive derivatives) were determined by spectrophotometric methods.

Results.The IMA levels in the diabetic patients were significantly higher than in the healthy controls and increased progressively with the degree of DN. The normoalbuminuria group had the lowest IMA level, which was signifi− cantly different from those in the micro− (p< 0.05) and macroalbuminuria (p< 0.01) groups. There were signifi− cant relationships between IMA and markers of DN (r= 0.47 for urinary albumin/creatinine and r= 0.36 for plas− ma creatinine) and oxidative stress (r= 0.38 for AOPPs and r= –0.44 for SH groups).

Conclusions.This study indicates that the observed increased IMA in T2DM is connected with diabetic nephropa− thy and oxidative protein damage. Measuring IMA plasma levels provided no additional information about nephropathy development, but it may be helpful in assessing the severity of oxidative stress, which causes kidney dysfunction (Adv Clin Exp Med 2009, 18, 4, 353–360).

Key words:type 2 diabetes mellitus, IMA, diabetic nephropathy, oxidative protein damage.

Streszczenie

Wprowadzenie.U chorych na cukrzycę oprócz przewlekłej hiperglikemii i nasilonego stresu oksydacyjnego występu− ją stany niedokrwienia i niedotlenienia, czego odzwierciedleniem jest zwiększone stężenie markerów oksydacyjnego uszkodzenia białek, szczególnie u osób z powikłaniami naczyniowymi, oraz albuminy modyfikowanej niedokrwieniem.

Cel pracy.Pomiar stężenia albuminy modyfikowanej niedotlenieniem (IMA) w osoczu pacjentów chorych na cu− krzycę typu 2 (T2DM) i z różnym stopniem nefropatii cukrzycowej (DN) oraz ocena związku między IMA, a ru− tynowymi markerami odzwierciedlającymi stopień niewydolności nerek (indeks albumina/kreatynia w moczu, stę− żenie kreatryniny w osoczu) oraz wskaźnikami oksydacyjnego uszkodzenia białek (zaawansowane produkty utle− niania białek (AOPP), grupy sufhydrylowe (SH) i karbonylowe (CO)).

Diabetes mellitus is not only a metabolic dis− order due to disturbances in insulin secretion and activity, but also a vascular disease. Metabolic abnormalities, which include hyperglycemia and abnormal carbohydrates, fats, and proteins han− dling, increase oxidative stress and activate the renin−angiotensin system. Type 2 diabetes mellitus (T2DM) is associated with increased incidence, severity, and delayed recovery from the ischaemic and hypoxic events that participate in disturbances of endothelial function and extracellular matrix remodeling and lead to the development of angiopathy, especially diabetic nephropathy [1–4]. Structural modifications of the albumin molecule can occur as a result of increased generation of reactive oxygen species (ROS). It has been sug− gested that a sensitive biomarker of cardiac ischemia, ischemia−modified albumin (IMA), is also an indicator of oxidative stress [5]. IMA increased before routine markers of acute myocar− dial infarction, such as cardiac troponin T and cre− atine kinase MB isoenzymes, and its level returned to baseline values in 6–12 h [5, 6]. It is suggested that the pathophysiological events of ischemia, including hypoxia, acidosis, and ROS generation, result in conformational changes of albumin. These changes alter the ability of the molecule to bind free metal ions such as cobalt, which became the basis for IMA determination by, for example, the albumin cobalt−binding test (ACB test®_{) [6, 7].}

The definitive and precise mechanism for IMA production in vivois still unknown. The N−termi− nus is most frequently mentioned as the binding site for free metal ions. However, an in vitrostudy under aerobic conditions showed that Co(II) binds preferentially to the metal binding sites A and B, not to the N−terminus of human albumin [8].

Recently, more and more studies are dealing with the role and diagnostic significance of IMA in different disorders of non−cardiac origin. Higher IMA levels were observed in physiological and pathological pregnancy, brain ischemia, infection, liver disease, trauma, some neoplasms, pulmonary embolism, peripheral vascular disease, and sys−

temic sclerosis and skeletal muscle ischemia [5, 9–11]. There is little information about IMA in renal diseases and the present authors have not found any papers concerning IMA in diabetic nephropathy (DN). Only Sharma et al. [12, 13] evaluated IMA in patients with myocardial ischemia and end−stage renal disease (ESRD) resulting from various diseases, including dia− betes, and referred for renal transplantation. They revealed IMA to be a moderately accurate marker of ischemia in these patients, but it can predict mortality.

In previous investigations the present authors found an approximately 75% higher value of IMA in patients with T2DM, especially those with coexisting micro− and macroangiopathy, in com− parison with healthy people. Such results suggest that albumin modification may occur in both the early late stages of the disease [14].

The aim of the present study was to examine plasma levels of IMA during nephropathy devel− opment in patients with type 2 diabetes mellitus and to determine their relationship with routine markers of kidney severity disease, i.e. the urinary albumin/creatinine ratio and plasma creatinine concentration as well as parameters of oxidative protein damage (OPD), such as the plasma levels of advanced oxidation protein products (AOPPs) and sufhydryl (SH) and carbonyl (CO) groups.

Material and Methods

Seventy−eight patients with types 2 diabetes mellitus treated at the Clinic of Angiology, Hypertension, and Diabetology of Wroclaw Medical University and 22 healthy adults (control group) were studied. None of the patients were known to suffer from an acute illness or inflam− matory condition at the time of the study. Sixteen individuals were treatment with oral hypo− glycemic agents, eleven with insulin, and fifty−one with combined therapy (oral hypoglycemic agents + insulin). In most cases (92%), the patients

Wyniki.Stężenie IMA u chorych na cukrzycę było istotnie wyższe niż w grupie kontrolnej i wzrastało progresyw− nie wraz z rozwojem stopnia DN. Najniższe stężenie IMA w grupie z normoalbuminurią było znacząco różne od wartości u pacjentów z mikroalbuminurią (p < 0,05) i makroalbuminurią (p < 0,01). Stwierdzono istotny związek między IMA a markerami DN (r = 0,47 z indeksem albumina/kreatynina oraz r = 0,36 ze stężeniem kreatyniny w osoczu), jak również z parametrami stresu oksydacyjnego (r = 0,38 z AOPP i r = –0,44 z grupami SH).

Wnioski.Wyniki badań własnych wskazują, że obserwowany w T2DM wzrost IMA jest związany ze stopniem ne− fropatii cukrzycowej oraz oksydacyjnym uszkodzeniem białek. Pomiar stężenia IMA w osoczu tych chorych nie dostarcza dodatkowych informacji na temat rozwoju nefropatii, ale może być pomocny w oszacowaniu intensyw− ności stresu oksydacyjnego, który powoduje uszkodzenie i zaburzenie czynności nerek (Adv Clin Exp Med 2009, 18, 4, 353–360).

received angiotensin−converting enzyme inhibi− tors (ACEIs). The healthy subjects did not have any inflammatory conditions, abnormalities in lipids and carbohydrates metabolism, or kidney disorders in routine medical checkups. All the par− ticipants were informed about the aim of this study and gave their consent to participate in it. The clin− ical and biochemical characterization of the stud− ied populations is given in Table 1.

Blood was drawn from the subjects of both groups in a fasting state into tubes containing heparin (250 units/ml) to obtain plasma samples. First morning samples of urine from the same patients were also taken. Both materials (plasma and urine) were frozen and stored at –80°C until further investigation, but for no longer than two months. The use of human blood and urine for research was approved by the Bioethics Committee of Wroclaw Medical University.

Ischemia−modified albumin (IMA) was deter−

mined by a manual colorimetric assay described by Bar−Or et al. [15] and called the Co(II)−albumin binding assay. This method consists of adding a known amount of exogenous Co(II) to a plasma sample and measuring unbound Co(II) colorimet− rically using dithiothreitol (DTT). The results are given in absorbance units (ABSU). The intra− and inter−assay coefficients for this manual technique were 2.31% and 4.21%, respectively, for the non− diabetic samples and 5.56% and 7.02%, respec− tively, for the diabetic ones.

In the same plasma samples the levels of para− meters reflecting oxidative damage of proteins were measured, i.e. advanced oxidation protein products (AOPPs) by the spectrophotometric method according to Witko−Sarsat et al. [16], sulfhydryl groups by the method with Ellman’s reagent [17], and carbonyl groups, as DNPH−reac− tive derivatives, by a 2,4−dinitrophenylhydrazine (DNPH) assay [18].

Parameters Control group Type 2 diabetes mellitus

(Wskaźniki) (Grupa kontrolna) (Cukrzyca typu 2)

Number – n) 22 78

(Liczba – n)

Sex – M/F 5/17 18/60

(Płec – M/K)

Age – years 55.20 ±16.20 62.10 ±13.10

(Wiek – lata)

Disease duration – years – 15.13 ±9.71

(Czas trwania choroby – lata)

Diastolic blood pressure – mmHg 72 ±19 80 ±20

(Ciśnienie rozkurczowe krwi – mmHg)

Systolic blood pressure – mmHg 118 ±29 141 ±14

(Ciśnienie skurczowe krwi – mmHg)

BMI (Indeks masy ciała) – kg/m2 _26.81 ±_{3.27 29.64} ±_4.81*

FPG (Stężenie glukozy w osoczu na czczo) – mmol/l 5.47 ±1.37 8.44 ±2.19***

HbA1c(Hemoglobina glikowana) – % 5.21 ±0.92 9.27 ±1.89***

Plasma albumin – g/l 51.11 ±5.54 54.01 ±6.91

(Stężenie albuminy w osoczu – g/l)

Total cholesterol – mg/dl 198.72 ±35.82 215,49 ±51.72

(Cakowity cholesterol – mg/dl)

HDL−cholesterol (Cholesterol HDL) – mg/dl 58.01 ±15.21 51.57 ±21.78 LDL−cholesterol (Cholesterol DL) – mg/dl 106.20 ±29.52 109.28 ±39.19

Triglycerides – mg/dl 102.70 ±48.51 186.04 ±93.48**

(Trójglicerydy – mg/dl)

Plasma creatinine – µmol/l 76.80 ±11.28 109.24 ±28.62*

(Stężenie kreatyniny w osoczu – µmol/l)

Urinary albumin/creatinine ratio – mg/g 21.00 ±13.50 219.50 ±98.60*** (Indeks albumina/kreatynina w osoczu – mg/g)

Table 1.The clinical and biochemical characteristic of the healthy controls and the patients with type 2 diabetes mellitus

Tabela 1.Kliniczna i biochemiczna charakterystyka grupy kontrolnej i chorych na cukrzycę typu 2

The routine Jaffe method was used for the plasma and urine concentrations of creatinine. The concentration of albumin in urine was determined according to Shosinsky et al. [19]. This allowed calculating the albumin/creatinine ratio (mg/g), a parameter frequently used in laboratory exami− nations [20]. Glycated hemoglobin (HbA1c) was

determined by a latex−enhanced turbidimetric immunoassay. Plasma glucose, albumin, total cho− lesterol and its fractions (HDL and LDL), and triglycerides were determined using routine clini− cal assays.

Date are expressed as mean values and the standard deviation. Statistical analysis was per− formed with Statistica PL for Windows, version 6.0. Statistical comparisons of the groups were made using the Mann−Whitney U test, Kolmo− gorov−Smirnov test, and/or ANOVA and the Kruskal−Wallis test. Correlation analysis was per− formed using Spearman’s test. Ap value of less than 0.05 was considered statistically significant.

Results

On the basis of the albumin/creatinine ratio, the diabetic patients were divided into three groups, with normoalbuminuria (< 30 mg/g, 37 patients), microalbuminuria (30–300 mg/g, 28 patients), and macroalbuminuria (> 300 mg/g, 13 patients). The plasma levels of IMA in these groups of patients with different degrees of renal failure in DN increased progressively together with the increase in albuminuria (Figure 1). The lowest level of IMA, i.e. that in the normoalbu− minuria group, was significantly different from those in the patients with microalbuminuria (p< 0.05) and macroalbuminuria (p< 0.01).

The levels of IMA and the markers of oxida− tive protein damage in the plasma of the healthy controls and all the patients with type 2 diabetes mellitus are given in Table 2. The values of all the examined parameters, except SH groups, were sig− nificantly (p < 0.001) higher in the diabetic patients than in the control group, by about 77% for IMA, 91% for AOPPs, and 39% for the DNPH−reactive derivatives (CO groups). The con− centration of the SH groups was significantly lower, by about 29% (p< 0.001), in the patients than in the healthy controls.

Fig. 1.Plasma levels of IMA in healthy controls and patients with type 2 diabetes mellitus (all) as well as in groups with normo−, micro−, and macroalbuminuria. Comparison with normoalbuminuria group. Statistical significance: *p < 0.05, **p < 0.01

Ryc. 1. Stężenie IMA w osoczu grupy kontrolnej i chorych na cukrzycę typu 2 oraz w grupie z nefropa− tią (normo−, mikro− i makroalbuminą). Porównanie z grupą z normoalbuminurią. Istotność statystyczna: *p < 0,05, **p < 0,01

Parameters Control group Type 2 diabetes mellitus (Wskaźniki) (Grupa kontrolna) (Cukrzyca typu 2) IMA (ABSU) 0.321 ±0.095 0.569 ±0.107***

AOPPs (µmol/l) 103.83 ±31.67 198.73 ±54.26***

SH groups (µmol/l) 632.05 ±89.10 450.20 ±70.00***

CO groups (nmol/l) 59.05 ±12.97 81.82 ±17.77***

Table 2.Ischemia−modified albumin and oxidative protein damage markers in plasma of the healthy controls and patients with type 2 diabetes mellitus

Tabela 2.Albumina modyfikowana niedokrwieniem i markery oksydacyjnego uszkodzenia białek w osoczu grupy kontrolnej i chorych na cukrzycę typu 2

In all the diabetic patients, the relationships between plasma level of IMA and the urinary albu− min/creatinine ratio, plasma creatinine concentra− tion, and the parameters of oxidation protein dam− age AOPP, SH, and CO were also determined, which is shown in Table 3. Plasma levels of IMA significantly correlated with the urinary albu− mina/creatinine ratio (r= 0.47, p< 0.01) and plas− ma creatinine concentration (r = 0.32, p < 0.05). Of the parameters of oxidative processes, signifi− cant (p < 0.05) relationships between IMA level and the concentrations of AOPPs and SH groups (r= 0.38 and r= –0.44, respectively) were found.

Discussion

Diabetic nephropathy is the most important microvascular complications in diabetic patients, especially those with T2DM. It is the most com− mon cause of renal failure, which develops over many years and leads to the development of ESRD, requiring hemodialysis and renal replace− ment therapy, which cause considerably decreased quality of life for these patients [21, 22]. Increasing evidence indicates that localized tissue oxidative stress and hypoxia are key components in the development of DN. On the other hand, pro− longed untreated diabetes mellitus leads to microangiopathy, tissue hypoxia, and ischemic lesions. It increases the risk for stroke and cardio− vascular and other tissue damage following ischemia [3, 4, 23, 24].

The main aim of the present cross−sectional study was to check whether the IMA level in the plasma of T2DM patients is connected with the stage of diabetic nephropathy, measured by the urinary albumin/creatinine ratio, as well as with the intensity of OS, reflected by parameters of

oxidative protein damage such as AOPPs and SH and CO groups, because IMA is also recognized as a marker of oxidative stress induced by different endovascular procedures, such as percutaneous coronary intervention [25].

In the present study the IMA plasma levels in the diabetic patients clustered into groups with normo−, micro−, and macroalbuminuria on the basis of the urinary albumin/creatinine ratio were measured. Albuminuria assessment is especially essential for diagnosing incipient nephropathy in diabetic patients, and the simplicity and low cost of its determination justify its preferential clinical use. A recent study indicates that the urinary albu− min concentration measured in 24−h urine collec− tion and the urinary albumin/creatinine ratio from spot morning urine had similar accuracy in screen− ing for microalbuminuria [26].

The present study found that IMA increased progressively with the degree of albuminuria. The lowest level, observed in patients with normoalbu− minuria, was significantly different from those in the microalbuminuria and macroalbuminuria groups, but there were also differences between patients with micro− and macroalbuminuria. Analyzing the relationships between IMA and parameters applied in the routine diagnosis of renal dysfunction, significant correlation with the urinary albumin/creatinine ratio and plasma creati− nine concentration as explanatory marker were found, as expected, but the relationship was stronger for the albumin/creatinine ratio.

To date, only Sharma et al. [12, 13, 27] have investigated plasma IMA level by the ACB test in a group of patients with ESRD, in whom diabetic nephropathy was established in about 40%. The authors indicated that the higher IMA level in these patients was probably of ischemic etiology. They concluded that IMA is a moderately accurate

Parameters IMA

(Wskaźniki) _{r p}

Plasma creatinine

(Kreatynina w osoczu) 0.36 0.0469

Urinary albumin/creatinine ratio

(Indeks albumina/kreatynina w moczu) 0.47 0.0081

Plasma levels AOPPs 0.38 0.0427

(Stężenie w osoczu) SH –0.44 0.0911

CO 0.20 0.4721

Table 3.Spearman’s rank correlation coefficients between plasma levels of IMA and markers of kidney function (plasma creatinine concentration and urinary albumin/creatinine ratio) as well as parameters of oxidation processes (AOPPs, SH, CO) in patients with type 2 diabetes mellitus

marker of myocardial ischemia, but a genuine marker of mortality and cardiac damage in patients with ESRD, with a sensitivity of 76% and speci− ficity of 74%. Classically, studies of the mecha− nisms underlying DN have focused on glomerular injury and the development of albuminuria. Increased urine albumin excretion is often accom− panied by rising blood pressure and cardiovascular risk in diabetic patients and increases its mortality [4, 21, 22].

In a previous study, the present authors found significantly higher plasma levels of IMA in T2DM patients and showed that its level should be considered in the context of well and poorly com− pensated diabetes, especially described by glycat− ed hemoglobin (HbA1c above 7%). Higher IMA

concentrations in diabetic patients with hyperten− sion as well as LDL hypercholesterolemia com− pared with baseline values were also found [14].

Free radicals are formed disproportionately in diabetes by glucose oxidation, nonenzymatic gly− cation of proteins, and the subsequent oxidative degradation of glycated proteins. As mentioned earlier, glycooxidation processes and disturbance of the oxidative−antioxidative balance commonly observed in diabetes can also induce chronic ischemia, which could lead to the necrosis of dif− ferent tissues and further accelerate the develop− ment of diabetic complications [22, 23, 28, 29]. For this reason, the present study also examined whether IMA is related to parameters of oxidation processes, such as AOPPs and SH and CO groups. They are regarded as good and useful markers reflecting the intensity of oxidative stress and as indicators of the severity of diabetic angiopathy [30–33]. To the present authors’ knowledge there is no correlation analysis in the literature between IMA and these biomarkers of oxidative protein damage. In the present study, significant relation− ships were found between plasma levels of IMA and AOPPs as well as SH groups, but not CO groups, in the diabetic patients.

In an earlier investigation by the present authors of markers of OPD, the largest change (highest increase) was observed for AOPPs [31]. That study also revealed that plasma AOPP level increased progressively with increasing albuminuria, which makes it useful in monitoring the development of diabetic nephropathy [33]. In vitrostudies showed that AOPPs, which accumulate in the diabetic kid−

ney, promote inflammation through the activation of renal NADPH oxidase. AOPPs are intensively formed during oxidative stress by the action not only of ROS, but also of chloramines produced by myeloperoxidase in activated neutrophils, especial− ly in an inflammatory state [16, 34].

Thiol groups are especially susceptible to oxidative stress and can create disulfide bonds or undergo transformation to carbonyl groups. They are capable of indirectly modifying other proteins. The latest one, recognized as uremic toxin, with a lower rate of degradation, can accumulate in dif− ferent tissues and accelerate the development of chronic renal failure [18, 32, 35]. Some authors indicate urinary CO and SH groups as markers of oxidative kidney damage in type 1 and 2 diabetic nephropathy [32, 36].

The present cross−sectional study, although somewhat limited due to its relatively small sam− ple size, indicated that increased plasma levels of IMA in T2DM is connected with diabetic nephropathy and with chronic oxidative stress and protein oxidation, which cause kidney dysfunc− tion. It is not yet known whether the conforma− tional changes of albumin are reversible or lead to preferential proteolytic degradation, as reported for other oxidized proteins [8, 37]. Some authors point to the large roles of oxidative stress as well as hypoxia in the development of diabetic distur− bances, for example in renal medullary hypoxia by hypoxia−inducible factors (HIFs) contributing to the development of DN [4]. Lately, hypoxia can be envisioned as a new mechanism that participates in insulin resistance in the adipose tissue of obese patients [38].

The present authors can conclude that deter− mining the plasma level of IMA in T2DM can pro− vide limited information about the development of this disease and its complications, but they suggest that it may be helpful in assessing oxidative stress during DN development. They hope that this study of the connection between IMA and parameters of kidney disease and markers of OPD in diabetes can also help investigators to consider antioxidants as proper agents in preventive medicine, especial− ly for preventing the development of diabetic com− plications and, perhaps, in antioxidant therapy. In the future it would be useful to widen these inves− tigations to persons with nephropathy but without diabetes.

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Address for correspondence:

Agnieszka Piwowar

Faculty of Pharmacy, Department of Biochemistry, Wroclaw Medical University

Szewska 38/39 50−139 Wrocław Poland

Tel.: +48 71 784 01 30

E−mail: [email protected]

Conflict of interest: None declared