Glycoxidation and inflammation in chronic haemodialysis patients

Marta Kalousová1,2, Sylvie Sulková3, Lenka Fialová1, Jirina Soukupová2, Ivan Matous Malbohan1,2, Pavel Spacek4, Martin Braun4, Ludmila Mikulíková2, Magdaléna Fortová3, Magdaléna Horejsí3, Vladimír Tesar5 and Tomás Zima2

1Institute of Medical Biochemistry, 2Institute of Clinical Chemistry, 3Department of Medicine Strahov, 4Institute of Rheumatology and 5First Department of Medicine–Division of Nephrology, First Faculty of Medicine, Charles University and General University Hospital, Prague, Czech Republic

Correspondence and offprint requests to: Marta Kalousová, MD, Institute of Medical Biochemistry, First Faculty of Medicine, Charles University, Katerinská 32, CZ 121 08 Prague 2, Czech Republic. Email: mkalousova{at}hotmail.com



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Uraemia and haemodialysis treatment are associated with microinflammation and oxidative as well as carbonyl stress, which result in enhanced formation of glycoxidation products. Although both glycoxidation and inflammation can contribute to severe vascular and cardiovascular complications, the role that these pathogenic mechanisms play in the complex response of the whole organism remains to be elucidated.

Methods. We performed a cross-sectional study in 34 clinically stable chronic haemodialysis patients and in 14 healthy controls while determining serum concentrations of pentosidine, fluorescent advanced glycation end-products (AGEs), advanced oxidation protein products (AOPPs) and acute phase reactants. We further assessed the relationship between these glycoxidation products and parameters of inflammation.

Results. Glycoxidation products as well as certain acute phase reactants were elevated in haemodialysis patients. There were significant correlations between AOPPs and inflammatory parameters such as orosomucoid (0.39, P < 0.05), fibrinogen (0.49, P < 0.05) and pregnancy-associated protein A (PAPP-A; 0.46, P < 0.05), but no correlations between pentosidine or fluorescent AGEs and any of the inflammatory parameters.

Conclusion. Oxidative damage showed a closer relationship to inflammation than advanced glycation (glycoxidation). AOPPs may represent a superior acute biochemical marker, whereas AGEs may better describe chronic long-lasting damage.

Keywords: advanced glycation end-products; advanced oxidation protein products; carbonyl stress; haemodialysis; inflammation; oxidative stress



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Both uraemia and treatment with haemodialysis are characterized by oxidative and carbonyl stress, microinflammation and various metabolic changes [1]. Oxidative and carbonyl stress lead to formation of glycoxidation products, including advanced glycation end-products [AGEs; the best known are carboxymethyllysine (CML) and pentosidine)], advanced oxidation protein products (AOPPs), advanced lipoperoxidation end-products (ALEs) and their precursors, which are reactive carbonyl species. Accumulation of these agents, with the help of carbohydrates and lipids, results in modification of proteins [2]. Microinflammation in haemodialysed patients is characterized by elevations in inflammatory markers and cytokines, such as C-reactive protein (CRP), serum amyloid A protein (SAA) and fibrinogen [3].

Apart from stimulating dyslipidaemia and calcium/phosphate metabolism disorders, oxidative and carbonyl stress can contribute to atherosclerosis and thereby lead to higher incidences of cardiovascular morbidity and mortality of haemodialysed patients [2,3]. AGEs exert several toxic effects. In addition to directly damaging tissue proteins, they are able to trigger the inflammatory response via interaction with their specific receptor (RAGE) and by causing activation of nuclear factor NF-{kappa}B. In vitro experiments have shown that they enhance transcription of genes for pro-inflammatory cytokines, growth factors and adhesive molecules [e.g. interleukin-6 (IL-6), tumour necrosis factor-{alpha} (TNF-{alpha}), granulocyte–macrophage colony-stimulating factor (GM-CSF), intercellular adhesion molecule (ICAM) and vascular cell adhesion molecule (VCAM)] [4], and other studies have shown that they may influence increased CRP production [1]. AOPPs, which are primarily formed from chlorinated oxidants by the action of myeloperoxidase, probably share several similar characteristics and biological activities with AGE-modified proteins [5,6]. Accelerated atherosclerosis is characterized by complement activation [7], decreased inhibition of complement due to glycation of its inhibitor [8], elevation of fibrinogen, an acute phase protein with prothrombotic effects, as well as oxidation- and glycation-induced elevations in CRP and modification of low-density lipoprotein (LDL). Recent studies have shown that CRP and other inflammatory markers, such as SAA and fibrinogen, are associated with cardiovascular morbidity and mortality as well as overall mortality [1,3].

Although both glycoxidation and inflammation have been associated with severe vascular and cardiovascular complications, the role that these pathogenic mechanisms play in the complex response of the whole organism remains to be determined. Although in vitro studies have shown direct influences of AGEs on cytokine production, in vivo experiments in patients have yielded controversial findings. Moreover, the latter were performed primarily with cytokines and CRP. The aim of the present study was to test the relationship between serum levels of prominent markers of glycoxidation and parameters of inflammation and acute phase reaction.



   Subjects and methods
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Patients and controls
We studied 34 chronic haemodialysis patients (17 men and 17 women), mean age 68 ± 12 years (43–89 years). Aetiology of renal failure was: diabetic nephropathy in six cases, interstitial nephritis in 11 cases, polycystic kidney disease in three cases, chronic glomerulonephritis in two cases, diabetic nephropathy and interstitial nephritis in three cases, diabetic nephropathy and vascular nephrosclerosis in three cases, interstitial nephritis and vascular nephrosclerosis in three cases, and multifactorial or unknown in three cases. At the time of the study, the patients had been treated with haemodialysis for 34 ± 44 months (3–198 months). They were dialysed three times a week for 4 h with a low-flux synthetic (polysulfone) membrane (F6, Fresenius Medical Care, Germany). All patients fulfilled the criteria of adequate dialysis (Kt/V >1.2 according to the Daugirdas II formula). Their serum albumin was 36 ± 3 g/l, haemoglobin was 93 ± 12 g/l, and they received a weekly average erythropoietin dose of 60 IU/kg body weight. The patients had serum concentrations of cholesterol and triglycerides within the normal range (exceptionally slightly above), and half of the patients were diabetic. Although a majority of patients were treated for hypertension, chronic ischaemic heart disease or both, there was no acute decompensation at the time of the study. The patients were in a stable clinical condition without signs of acute infection, were not treated with antioxidants and all gave informed consent prior to entering the study.

The control group consisted of 14 healthy subjects (seven men and seven women), mean age 61 ± 8 years (53–77 years). They were not taking antioxidants and they gave informed consent prior to entering the study.

The study was approved by the local Institutional Ethical committee.

Blood samples
Blood samples from haemodialysed patients were collected via the arteriovenous fistula before starting the dialysis session. Samples from controls were obtained via venepuncture of the cubital vein. Blood was centrifuged at 1450 g (4°C) for 10 min. Serum was stored at -80°C and processed within 3 months.

Pentosidine assay
Pentosidine was determined by reversed-phase high-performance liquid chromatography (HPLC) according to Spacek and Adam [9]. Briefly, samples were mixed with equal amounts of 35% HCl and hydrolysed for 16 h at 105°C. The hydrolysate was mixed with a suspension of cellulose combined with a solution containing n-butyl alcohol and acetic acid, and this mixture was then applied to a purification column filled with cellulose. The pentosidine-containing fraction was then desorbed with 0.05 mol/l HCl and the extract was evaporated. The residue was reconstituted in a solution containing 0.02 mol/l heptafluorobutyric acid, 0.01 mol/l ammonium sulfate and acetonitrile (this mixture was also used as the mobile phase) and this was applied to an HPLC system (Shimadzu) with a C18 reversed-phase column. We monitored the emission signal at 385 nm upon excitation at 335 nm. Synthetic pentosidine was used as a standard. The concentration of pentosidine was expressed in nmol/l and nmol/g protein.

AGEs assay
Determination of fluorescent AGEs is based on spectrofluorimetric detection according to Henle [10] and Munch [11]. Serum was diluted 1:50 with PBS (phosphate-buffered saline) pH 7.4, and fluorescence intensity was recorded in emission maximum (~440 nm) upon excitation at 350 nm (Fluoromax-3 spectrofluorometer, Jobin Yvon Horiba, USA). Fluorescence intensity was expressed in arbitrary units as AU and as AU/g protein.

AOPP assay
AOPP determination was based on spectrophotometric detection according to Witko-Sarsat [5]. For calibration, we applied 200 µl of serum diluted 1:5 with PBS pH 7.4, 200 µl of chloramine T (0–100 µmol/l) and 200 µl of PBS as blank to a microtitre plate. We then added 10 µl of 1.16 mol/l potassium iodide and 20 µl of acetic acid, and absorbance at 340 nm was measured immediately (Multiskan Ascent photometer, Labsystems, Finland). Concentrations of AOPP, determined in reference to the calibration, were expressed in µmol/l.

Determination of inflammatory markers
Inflammation markers, such as CRP, complement factors C3 and C4, {alpha}2-macroglobulin, orosomucoid and fibrinogen, were determined by standard clinical chemistry methods, as recommended by the IFCC (CRP by turbidimetry and other markers by nephelometry, Image, Beckman Coulter, USA). SAA was determined using a standard enzyme-linked immunosorbent assay (ELISA) kit (Biosource, USA). Pregnancy-associated protein A (PAPP-A) was assessed immunochemically with the TRACE (time-resolved amplified cryptate emission) method (Kryptor, Brahms, Germany and standard kits Cezanne, France).

Statistics
All results are expressed as medians and interquartile ranges. Differences between groups were evaluated using Mann–Whitney U-tests. The correlation coefficient r (Pearson, Spearman) was used to examine relationships between parameters. Differences were considered statistically significant at P < 0.05.



   Results
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
We found that glycoxidation parameters (pentosidine, AGEs and AOPPs) as well as some acute phase reactants (CRP, orosomucoid, fibrinogen and PAPP-A) were elevated in haemodialysed patients compared with healthy subjects. These findings are shown in Table 1.


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Table 1. Serum concentrations of glycoxidation products and markers of inflammation in chronic haemodialysis patients

 
AOPP was significantly correlated with orosomucoid (0.39, P<0.05), fibrinogen (0.49, P<0.05) and PAPP-A (0.46, P<0.05) in haemodialysed patients (Figure 1). However, AOPP was not correlated with the other inflammatory markers, and there were no correlations of pentosidine or fluorescent AGEs with any of the inflammatory parameters.



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Fig. 1. Correlation between AOPP and PAPP-A in chronic haemodialysis patients. r = 0.46, P < 0.05. y = -2.785 + 0.18656 x x.

 


   Discussion
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
It has long been recognized that haemodialysed patients experience elevations in AGEs as well as some inflammatory markers. In vitro studies have shown that AGEs can act via their specific receptor to produce increased production of proinflammatory cytokines. Moreover, AGEs may also form at sites of inflammation by using the myeloperoxidase pathway [12]. These findings may explain the previously described relationship between glycation and inflammation markers. Nevertheless, examinations of serum/plasma in patients with renal insufficiency have yielded controversial results. For example, Sebeková et al. [13] failed to detect any correlations between fluorescent AGEs and CML with IL-6 or TNF-{alpha} in children with chronic renal insufficiency, in haemodialysed or in transplanted patients, whereas in vivo studies from Witko-Sarsat et al. [5,6] have provided support for this relationship.

We failed to detect relationships between classical markers of inflammation or acute phase reactants in serum with pentosidine and other fluorescent AGEs. This finding is similar to that found for CRP and CML [13,14]. However, a relationship between CRP, which is a risk factor for cardiovascular diseases in haemodialysed patients [3], and advanced glycoxidation products has often been proposed. In addition, others have suggested a direct influence of these products on CRP production [1]. In contrast, Miyata et al. [15] found a significant correlation between pentosidine and CRP in rheumatoid arthritis patients, who have more pronounced chronic inflammation than haemodialysed patients.

In haemodialysis patients, both AOPPs and AGEs are elevated, and these products share several similar characteristic features with AGE-modified proteins, such as formation via oxidative stress and similar biological effects. They have been described as an independent risk factor for coronary artery disease, in part because their plasma levels are significantly related to coronary artery pathology [16]. Despite similarities between AOPPs and AGEs, there are slight differences between these products, such as their relationship to markers of inflammation in serum and the finding that AGEs decrease, whereas AOPPs increase during dialysis with diacetate cellulosic membranes [17]. Other differences include the higher potency of AOPPs in triggering monocyte respiratory bursts [6]. AOPPs correlate well with certain cytokines [5,6] as well as with some markers of inflammation, including fibrinogen, orosomucoid and PAPP-A (which is a new marker that may also act as an acute phase reactant). Because of its prothrombotic effects, fibrinogen is closely related to atherosclerosis, and urinary excretion of orosomucoid was shown to be a predictor of mortality in patients with diabetes mellitus type 2 [18]. Although we failed to detect a correlation between CRP and AOPPs, other studies reported that CRP correlates well with other markers of oxidative stress, such as F2-isoprostanes and lipid peroxidation products.

PAPP-A has been routinely assayed for prenatal screening of Downs syndrome in the first trimester of pregnancy. It was later discovered that PAPP-A is the insulin-like growth factor (IGF)-dependent IGF-binding protein-4 protease that is secreted by human fibroblasts [19]. Its levels in pregnant women are ~1000-fold higher than in non-pregnant women. Nevertheless, PAPP-A can be detected in very low amounts even in healthy individuals (both non- pregnant women and men), and its slight elevation can serve as a new and exquisite marker of acute coronary syndrome and may predict cardiovascular risk better than CRP [20]. PAPP-A is expressed abundantly in unstable plaques from patients with angina pectoris, but its expression in stable atherosclerotic plaques is only minimal [20]. Slight elevations in PAPP-A, such as observed during acute myocardial infarction, are also typical for haemodialysed patients. Further investigations will be necessary to determine whether there is a causal relationship between PAPP-A and AOPPs.

In conclusion, the present findings indicate that results from clinical chemistry examinations can differ from studies performed in vitro because of the complexity of reactions occuring in intact organisms. There are slight differences between AOPPs, which act as a pure oxidative stress marker, and AGEs, which behave as both oxidative and carbonyl stress markers. AOPPs show a closer relationship to inflammation and acute phase reaction than AGEs. Because of the relationship of AOPPs with formation of inflammation, their relationship with certain inflammatory markers, their higher potency in triggering respiratory bursts and changes during the haemodialysis sessions, AOPPs may provide a superior acute marker, whereas AGEs may represent a better marker of chronic long-lasting damage.



   Acknowledgments
 
The authors are grateful to Mrs Kremenová, Mrs Miskovská and Mrs Kucharová for technical assistance, and to Ing. Míková for statistical evaluation. This study was supported by grant IGA MH CZ No NB/7035-3.

Conflict of interest statement. None declared.



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 Discussion
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Received for publication: 10.10.02
Accepted in revised form: 18. 4.03





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