Increased levels of N{epsilon}-(carboxymethyl)lysine and N{epsilon}-(carboxyethyl)lysine in type 1 diabetic patients with impaired renal function: correlation with markers of endothelial dysfunction

Mariska L. M. Lieuw-A-Fa1,3, Victor W. M. van Hinsbergh1,3,6, Tom Teerlink2, Rob Barto4, Jos Twisk3,5, Coen D. A. Stehouwer3,4 and Casper G. Schalkwijk2,3

1Department of Physiology, 2Department of Clinical Chemistry, 3Institute of Cardiovascular Research, 4Department of Internal Medicine and 5Department of Clinical Epidemiology and Biostatistics, Vrije Universiteit Medical Centre, Amsterdam and 6Gaubius Laboratory TNO-PG, Leiden, The Netherlands

Correspondence and offprint requests to: Dr C. G. Schalkwijk, Department of Clinical Chemistry, Vrije Universiteit Medical Center, PO Box 7057, 1007 Mbyte Amsterdam, The Netherlands. Email: C.Schalkwijk{at}vumc.nl



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Diabetic and non-diabetic patients with renal failure have an increased risk for cardiovascular disease, which may be the result of uraemic toxins, including advanced glycation end-products (AGEs). The aim of the study was to investigate the levels of well-characterized AGEs, N{epsilon}-(carboxymethyl)lysine (CML) and N{epsilon}-(carboxyethyl)lysine (CEL) in relation to kidney function and to study the relationship of these AGEs to endothelial function and inflammation in type 1 diabetic patients.

Methods. Plasma levels of CML and CEL were measured in 60 type 1 diabetic patients categorized as having normal glomerular filtration rate (GFR) (>80 ml/min, n = 31) or decreased GFR (<80 ml/min, n = 29) as estimated by the Cockcroft–Gault formula. To assess the relationship of these AGEs to endothelial function and inflammation, markers of endothelial function von Willebrand factor (vWf), soluble vascular cellular adhesion molecule-1 (sVCAM-1), soluble E-selectin (sE-selectin), soluble thrombomodulin (sTM), tissue type-specific plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1), and C-reactive protein (CRP), a marker of inflammatory activity, were determined by enzyme-linked immunosorbent assays.

Results. Plasma levels of CML and CEL were increased in diabetic patients with decreased GFR as compared with patients with normal GFR [CML 4.9 (2–12.6) vs 2.9 (1.7–4.4) µmol/l, P<0.000; and CEL 1.7 (0.9–3.3) vs 1.2 (1.7–4.4) µmol/l, P = 0.004, respectively). Independently of the GFR, the plasma levels of CML and CEL were significantly associated with sVCAM-1, vWf and sTM.

Conclusions. Plasma levels of CML and CEL rise with deterioration of GFR. Furthermore, CML and CEL levels are associated with markers of endothelial activation independently of renal function. This suggests an involvement of these AGEs in the acceleration of cardiovascular complications in patients with renal impairment.

Keywords: advanced glycation end-products; endothelial markers; renal impairment



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Diabetic patients with end-stage renal failure, as well as those with mild renal impairment, have an increased risk for cardiovascular disease, the aetiology of which remains incompletely understood [1,2]. This high risk has been attributed to risk factors associated with uraemia such as dyslipidaemia and increased oxidative stress. In addition to these traditional risk factors, increased levels of advanced glycation end-products (AGEs) may also contribute to the increased risk for vascular disease [3].

AGEs are a heterogeneous family of compounds derived from the reaction of reducing sugars including glucose with amino groups in proteins through a series of oxidative and non-oxidative reactions. N{epsilon}-(carboxymethyl)lysine (CML) has been identified as the major non-fluorescent AGE in vivo [4]. Next to glucose, reactive di-carbonyl compounds such as methylglyoxal are major precursors in the formation of cellular and extracellular AGEs [5]. Methylglyoxal can react with arginine residues to form imidazolone adducts and with lysine residues to form N{epsilon}-(carboxyethyl)lysine (CEL). AGEs accumulate in diabetes mellitus and tend to rise even further in diabetes when diabetic nephropathy ensues [6]. In uraemia, in the absence of diabetes, a 2- to 3-fold increase in AGEs has been reported, which indicates that the kidney plays an important role in the accumulation of these compounds [7].

Therefore, the first aim of this study was to examine the relationship between AGEs and renal function in a group of type 1 diabetic patients, who were divided into those with a normal glomerular filtration rate (GFR) (>80 ml/min) or decreased GFR (<80 ml/min). We focused on two important AGEs, namely CML and CEL.

There is ample evidence of the involvement of CML and methylglyoxal-derived AGEs in the acceleration of cardiovascular disease [8,9]. Since deterioration of renal function is associated with an acceleration of vascular pathology and accumulation of AGEs, it is of particular interest to study whether the accumulation of these AGEs is associated with markers of endothelial function and inflammation, as both endothelial function and inflammation have a central role in the pathogenesis of vascular diseases [10]. The second aim of the study was therefore to investigate the relationship of these AGEs to endothelial function and chronic inflammation, two important determinants of vascular disease. We measured von Willebrand factor (vWf), soluble vascular cellular adhesion molecule-1 (sVCAM-1), soluble E-selectin (sE-selectin), soluble thrombomodulin (sTM), tissue type-specific plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) as markers of endothelial function, and C-reactive protein (CRP) as a marker of inflammatory activity.



   Subjects and methods
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Subjects
Biochemical parameters were determined in plasma of 60 patients with type 1 diabetes. This population has been described before [11]. In these patients, no overt signs of infection were present, nor were any patients treated by dialysis. Patients were categorized into two groups based on GFR into normal (>80 ml/min) or decreased GFR (<80 ml/min). The creatinine clearance was calculated according to the Cockcroft–Gault formula: [140 – age (years) x weight (kg) x F]/[plasma creatinine (µmol/l) x 72] in ml/min. F =1 if male and 0.85 if female. The Cockcroft–Gault formula is expressed per 1.73 m2 body surface area [12]. The median albumin excretion rate (AER) was measured in three 24 h urine samples. HbA1c was measured with high-performance liquid chromatography (HPLC; normal range 4.3–6.1%).

Detection of CML and CEL by LC/MS/MS
Plasma levels of CML and CEL were measured by stable-isotope dilution tandem mass spectrometry. To 25 µl of plasma, 500 µl 100 mmol/l sodium borohydride, dissolved in borate buffer (200 mmol/l; pH 9.2) was added. After 4 h at room temperature, proteins were precipitated with trichloroacetic acid and pelleted by centrifugation. After addition of [2H4]CML and [2H8]CEL as internal standards (kindly provided by Drs J. Baynes and S. Thorpe, University of South Carolina, Columbia), proteins were hydrolysed overnight with 6 mol/l HCl at 110°C. HCl was evaporated under a stream of nitrogen at 80°C and the residue was dissolved in 5 mM nonafluoropentanoic acid (NFPA) and analysed by reversed-phase HPLC on a Symmetry C18 column (3.9 x 150 mm; 5 µm particle size; Waters, Milford, MA) using NFPA as ion-pair reagent. Mobile phase A consisted of 5 mM NFPA and mobile phase B was acetonitrile. Compounds were eluted with a linear gradient from 10 to 25% B in 10 min. Thereafter, strongly retained compounds were removed from the column by a strong solvent wash: between 10 and 12 min, %B was increased to 80%, kept at this percentage until 14 min and, between 14 and 16 min, %B was reduced to 10% and kept at this percentage to equilibrate the column before the next injection at 21 min. Flow rate was 1 ml/min and 20% of the column effluent was introduced into the turbo-ionspray source of the tandem mass spectrometer (API 3000 from Sciex/Applied Biosystems). Analyses were performed in the positive ion mode. Multiple reaction monitoring was performed for the transitions 205.1/84.1 and 219.1/84.1 for CML and CEL, respectively, and 209.1/88.1 and 227.1/92.1 for the internal standards [2H4]CML and [2H8]CEL, respectively. CML, CEL and the internal standards eluted as symmetrical peaks and were clearly resolved from interfering compounds. Typical mass chromatograms of a plasma sample are shown in Figure 1. Mean recoveries of exogenous CML added to plasma in concentrations of 0.25 and 0.50 µmol/l were 100 and 95%, respectively. Mean recoveries for CEL at these concentrations were 92 and 94%, respectively. Within-day and between-day CVs were <4.4% and <3.2% for CML and <6.8% and <7.3% for CEL. Mean plasma levels of CML and CEL (±SD) in 10 healthy individuals were 2.8±0.4 and 0.8±0.2 µmol/l, respectively.



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Fig. 1. LC/MS/MS of a plasma sample using multiple reaction monitoring in positive ion mode. From top to bottom, mass chromatograms of the following transitions are shown: 205.1/84.1 for CML; 209.1/88.1 for [2H4]CML; 219.1/84.1 for CEL; 227.1/92.1 for [2H8]CEL.

 
Measurements of endothelial function and inflammation
Plasma vWf antigen was measured by an enzyme-linked immunosorbent assay (ELISA), using rabbit anti-vWf antigen as a catching antibody and a peroxidase-conjugated rabbit anti-vWf antigen as a detecting antibody (Dako, Copenhagen, Denmark). The concentration of vWf is expressed as the percentage of antigen concentrations in normal pooled plasma, which is defined as 100%. The intra- and interassay variations are 2.3 and 3.8%, respectively. Commercially available ELISA kits were used for the measurement of tPA antigen (Imulyse tPA, Biopool, Umeå, Sweden), PAI-1 (Innotest PAI-1, Innogenetics, Zwijndrecht, Belgium), sE-selectin (R&D Systems, Minneapolis, USA), sVCAM-1(Diaclone, Besancon, France) and sTM (Diagnostica Stago Asniéres, France). All measurements were performed in duplicate, and with intra- and interassay coefficients of variation <10%.

CRP was measured with a highly sensitive in-house ELISA with rabbit anti-CRP (Dako, Copenhagen, Denmark) as a catching antibody and a horseradish peroxidase (HRP)-conjugated rabbit anti-CRP tagging antibody, with intra- and interassay coefficients of variation of 3.8 and 4.7%, respectively.

Statistical analysis
CML and CEL, creatinine, AER, CRP, tPA, PAI-1, sTM, sVCAM-1 and sE-selectin required a log transformation to satisfy the statistical requirement of normality. First of all, differences between groups were assessed by means of an independent sample t-test for the normally distributed and log-transformated data. The differences for vWf and HbA1c between the groups were assessed by the {chi}2 test.

Secondly, the influence of renal function, defined as GFR, on the AGE levels was examined. Univariate regression analysis was used to assess the relationship between GFR and AGE levels with the individual AGE levels as dependent variables in the model. To evaluate the influence of several possible co-variates [i.e. age, sex, CRP, HbA1c, diabetes duration, body mass index (BMI), systolic and diastolic blood pressure, use of anti-hypertensives or an angiotensin-converting enzyme (ACE) inhibitor] on the relationship between the AGEs and renal function, a multiple linear regression analysis was performed in which these co-variates were added to the model as independent variables. Thirdly, to assess the relationship between CML and CEL with markers of endothelial activation and a marker of low grade chronic inflammation (CRP), univariate analysis was applied with the endothelial markers and CRP as dependent variables. To evaluate the influence of renal function on the relationship between CML and CEL with markers of endothelial activation and CRP, multiple linear regression was applied in which GFR was added to the regression model as an independent variable. To exclude the influence of other confounding factors on the relationship between markers of endothelial function and CRP to AGEs, age, sex, CRP, HbA1c, diabetes duration, BMI, systolic and diastolic blood pressure, use of anti-hypertensives or ACE inhibitors were also added to the model as independent variables. Statistical analysis was performed using SPSS 9.0 (SPSS Inc., Chicago, IL). P<0.05 indicates a statistically significant difference.



   Results
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
The characteristics of the type 1 diabetic patients categorized as having normal or decreased GFR are shown in Table 1. Age, diabetes duration and BMI differed between the groups, whereas HbA1c, systolic blood pressure and diastolic blood pressure did not differ.


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Table 1. Characteristics of 60 type 1 diabetic patients

 
Reference values for CML and CEL were obtained in 10 healthy individuals and were 2.8 (2.1–3.4) and 0.8 (0.5–1.2) µmol/l for CML and CEL, respectively. There was a significant correlation between plasma levels of CML and CEL (r = 0.622, P<0.0001). In type 1 diabetic patients with normal renal function, CEL levels [1.2 (0.7–2.4) µmol/l], but not CML levels [2.9 (1.7–4.4) µmol/l], are significantly (P<0.005) increased, as compared with our control group. In type 1 diabetic patients with impaired renal function, plasma levels of CML and CEL are increased (1.7- and 1.4-fold, respectively), as compared with patients with normal GFR (Table 1). After adding GFR and AER as co-variates to the univariate analysis, the increase of CML was associated with GFR instead of with AER (data not shown).

Table 2 shows the influence of renal function, as defined by GFR, on plasma levels of CML and CEL. There was a significant association of CML and CEL with GFR. For CML, the relationship with GFR remained strong and highly significant after adjusting for age, sex, diabetes duration, HbA1c, BMI, systolic and diastolic blood pressure, use of antihypertensives, use of ACE inhibitors and CRP. The association between CEL and GFR remained strong after adding the other co-variates to the model, but was no longer significant.


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Table 2. Crude and adjusted standardized regression coefficients for the relationship between levels of CML and CEL (as dependent variables) and GFR as estimated by the Cockcroft–Gault formula (independent variable)

 
Statistically significant associations between the two AGEs and sVCAM-1, sTM, vWf and CRP were found (Table 3). After adding GFR to the model, the positive correlation between CML and sVCAM-1 remained strong and highly significant. The relationship between CML and sTM was affected by adding GFR to the model but remained highly significant. Adding other co-variates to the model changed the relationship of CML to sVCAM-1 and sTM, but it remained significant. In contrast, the relationships of CML to CRP and vWf was no longer significant after adding GFR to the model.


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Table 3. Crude and adjusted standardized regression coefficients for the relationship between sVCAM-1, sTM, vWf and CRP (dependent variables) and CML and CEL (independent variables)

 
The associations between CEL and sVCAM-1, sTM and vWf were reduced after adding GFR to the model but remained significant. In contrast, the relationship between CEL and CRP was no longer significant after adjusting for GFR. The association between CEL and sVCAM-1 was no longer significant after adjusting for the other co-variates.



   Discussion
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
This study shows an increase in plasma levels of CML and CEL in type 1 diabetic patients with decreased GFR as compared with patients with a normal GFR. The CML and CEL levels were positively correlated with sVCAM-1, sTM and vWF, markers of endothelial function, and CRP, a marker of inflammation. The associations of CML with sVCAM-1 and sTM on the one hand and CEL with vWf and sTM on the other, remained significant after adjusting for GFR.

Numerous studies have focused on the effect of kidney function on AGE levels [6,13,14]. In agreement with a study done in type 2 diabetic patients, we found that CML levels did not differ between control subjects and subjects with type 1 diabetes in the presence of normal kidney function, but CML levels increased with impaired kidney function [13]. We showed that CML concentrations were influenced by glomerular filtration independently of other potential confounders.

CEL levels also increased with impaired renal function. This is in agreement with a study done by Degenhardt et al. who found an increase in plasma CEL in patients with uraemia in comparison with control non-diabetic subjects [14]. In addition, we show that CEL levels were elevated in diabetic patients with normal GFR as compared with healthy controls and further increased when GFR decreases, implying a role for both diabetes and renal function in the determination of plasma levels of CEL.

Renal function impairment has been implicated as an important determinant of cardiovascular disease [1]. The clinical importance of the relationship between increased AGE levels and impaired renal function may be the biological activities of these AGEs, which lead to endothelial dysfunction and inflammatory activation of the vascular wall, two phenomena that are involved in vascular disease. VCAM-1 is implicated in the pathogenesis of atherosclerosis and sVCAM-1 is thought to be mainly derived from the activated vessel wall [15]. This association may be an indication of a role for AGEs as uraemic toxins for endothelial cells [16].

Our results show that the association of sVCAM-1 with CML and CEL levels is independent of GFR. After adjustment for other possible confounders, the relationship between CML and sVCAM-1 was the only association that remained significant. This may indicate a direct effect of CML on the endothelium, as has been implied [8]. It has been reported in in vitro and in vivo studies that CML-modified proteins can increase the expression of VCAM-1 on endothelial cells after ligation to their receptor RAGE. Furthermore, it was reported that these CML-modified proteins increase monocyte adhesion [8]. Both processes, VCAM-1 expression and monocyte adhesion, are crucial to plaque development [10]. These data suggest a role for CML-modified proteins in the pathogenesis of cardiovascular diseases.

Another possibility that must be considered is that a common process, such as an enhanced oxidative stress status, could lead to both CML formation and endothelial activation. Although CML is generally regarded as a glycoxidation product, it can also be formed through lipid peroxidation and the generation of glycolaldehyde via the myeloperoxidase pathway [17,18]. Indeed, CML cannot be formed without oxidative stress [4]. A role for oxidative stress has been suggested in children with chronic renal failure in whom, after successful renal transplantation, fluorescent AGE levels decreased, but CML levels remained elevated. It was speculated that this was due to enhanced oxidative stress in these patients [19]. Therefore, we cannot exclude that a common denominator, such as oxidative stress, contributes to both the generation of AGEs and endothelial activation, and may act as a confounder in the association between CML and sVCAM-1.

The association of CML and CEL with sTM that we found remained significant after adjusting for GFR and other confounders. The exact role of sTM has not been fully elucidated, but elevated levels were found in various diseases, such as atherosclerosis [20]. It is known that sTM levels are related to kidney function. To distinguish whether the sTM elevation was due to endothelial damage or to increased sTM due to renal failure, the sTM levels may be expressed as a ratio of sTM/serum creatinine [20]. In doing so, we found no differences between the two GFR groups, indicating that correcting for renal function abrogated the differences in sTM between the two groups. This suggests that release of sTM from the endothelium is not a major determinant for the variation in sTM levels observed among these groups.

The associations between CML on the one hand and CRP and vWf on the other were explained by impaired renal function. CEL levels showed a weak but significant association with vWf after correcting for GFR and other confounders, but no relationship with CRP levels.

In summary, this study shows that CML and CEL levels are increased in type 1 diabetic patients with decreased GFR as compared with patients with normal GFR, irrespective of albuminuria status. It also shows that the association between CML and CEL with markers of endothelial activation, sVCAM-1, vWf and sTM, is independent of kidney function. Although this is a study done in a small number of subjects and does not allow a conclusion on causality, these data may suggest a role for CML and CEL in the activation of the endothelium and development of cardiovascular diseases in patients with renal impairment, at least in type 1 diabetes.



   Acknowledgments
 
This study was supported by a grant from the Diabetes Fonds Nederland (C.S.), a University Stimulating Fund grant (V.v.H.) and the Dutch Kidney Foundation.

Conflict of interest statement. None declared.



   References
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 20. 3.03
Accepted in revised form: 22.10.03