Profound Mishandling of Protein Glycation Degradation ...€¦ · clearance in chronic renal...

Profound Mishandling of Protein Glycation DegradationProducts in Uremia and Dialysis

Stamatina Agalou,* Naila Ahmed,* Roya Babaei-Jadidi,* Anne Dawnay,† andPaul J. Thornalley**Department of Biological Sciences, University of Essex, Colchester, Essex; †Renal Research Laboratory, St. Bartholomew’sand The Royal London School of Medicine and Dentistry, St. Bartholomew’s Hospital, London, United Kingdom

The aim of this study was to define the severe deficits of protein glycation adduct clearance in chronic renal failure andelimination in peritoneal dialysis (PD) and hemodialysis (HD) therapy using a liquid chromatography-triple quadrupole massspectrometric detection method. Physiologic proteolysis of proteins damaged by glycation, oxidation, and nitration formsprotein glycation, oxidation, and nitration free adducts that are released into plasma for urinary excretion. Inefficientelimination of these free adducts in uremia may lead to their accumulation. Patients with mild uremic chronic renal failurehad plasma glycation free adduct concentrations increased up to five-fold associated with a decline in renal clearance. Inpatients with ESRD, plasma glycation free adducts were increased up to 18-fold on PD and up to 40-fold on HD. Glycationfree adduct concentrations in peritoneal dialysate increased over 2- to 12-h dwell time, exceeding the plasma levels markedly.Plasma glycation free adducts equilibrated rapidly with dialysate of HD patients, with both plasma and dialysate concentra-tions decreasing during a 4-h dialysis session. It is concluded that there are severe deficits of protein glycation free adductclearance in chronic renal failure and in ESRD on PD and HD therapy.

J Am Soc Nephrol 16: 1471–1485, 2005. doi: 10.1681/ASN.2004080635

B oth peritoneal dialysis (PD) and hemodialysis (HD)therapy as practiced currently have high mortality andmorbidity—the median survival time from commence-

ment of dialysis therapy is 5 to 8 yr, depending on otherexacerbating factors (old age and diabetes). Mortality and mor-bidity are associated with an increased risk for cardiovasculardisease. Inadequate removal of uremic toxins is a primarycause of uremia associated vascular disease (1). Protein glyca-tion adducts are a class of uremic toxin (2), but it is currentlyunclear how effective conventional HD and PD therapies are inremoving glycation adducts.

Glycation of proteins is a complex series of parallel andsequential reactions collectively called the Maillard reaction. Itoccurs in all tissues and body fluids. Early stage reactions withglucose lead to the formation of the early glycation adductfructosyl-lysine (FL), and later stage reactions form advancedglycation end products (AGE) (3). FL degrades slowly to formAGE. Glyoxal (G), methylglyoxal (MG), and 3-deoxyglucosone(3-DG) are also potent glycating agents that are formed by thedegradation of glycated proteins, glycolytic intermediates, andlipid peroxidation. They react with proteins to form AGE di-rectly (Figure 1, a and b). Important AGE quantitatively arehydroimidazolones derived from arginine residues modifiedby glyoxal, MG and 3-DG, G-H1, MG-H1, and 3DG-H, respec-

tively (Figure 1c). Other important and widely studied AGEare N�-carboxymethyl-lysine (CML), N�-carboxyethyl-lysine(CEL), and pentosidine (4) (Figure 1, d and e). Proteins alsosuffer oxidative and nitrosative damage forming methioninesulfoxide (MetSO) (5) and 3-nitrotyrosine (3-NT) (6) (Figure 1f).Glycation adduct residues are formed by the physiologic gly-cation of endogenous cellular and extracellular proteins and arealso present in ingested food (7). Glycation free adducts arefound in plasma, urine, and other physiologic fluids. Theyoriginate from the turnover of endogenous glycated proteins bycellular proteolysis and from food (4,8). Glycation reactions areincreased in uremia, where there are increased concentrationsof many �-oxoaldehydes, particularly glyoxal, MG, and 3-DG(9,10). �-Oxoaldehydes are also present in PD fluids, formed bythermal sterilization (11), and introduced into the peritonealcavity, they add to the increased glycation potential in ESRD.

The normal high renal clearance of AGE free adducts (4) isimpaired markedly in ESRD; hence, the concentrations of freeAGE in plasma are expected to increase. In this study, weevaluated the characteristics of glycation free adduct excretionof patients who had chronic renal failure (CRF) with milduremia and patients who had ESRD with severe uremia andwere on HD and PD therapies.

Materials and MethodsClinical Sampling: Patient Characteristics and Blood andUrine Sample Collection

Venous blood samples were drawn from normal healthy subjects,untreated CRF patients, and ESRD patients into tubes with heparinanticoagulant. Subject characteristics are given in Table 1. Renal creat-inine clearance for all dialysis patients was �10 ml/min. Blood samples

Received August 4, 2004. Accepted February 16, 2005.

Published online ahead of print. Publication date available at www.jasn.org.

Address correspondence to: Dr. Paul J. Thornalley, Department of BiologicalSciences, University of Essex, Wivenhoe Park, Colchester, Essex CO4 3SQ, UK.Phone/Fax: �44-1206-873010; E-mail: [email protected]

Copyright © 2005 by the American Society of Nephrology ISSN: 1046-6673/1605-1471

Figure 1. Protein glycation in physiologic systems. (a) Pathways for the formation of advanced glycation endproducts (AGE). (b)�-Oxoaldehyde glycating agents. (c) Hydroimidazolone AGE: glyoxal-H1 (G-H1; 214 Da), methylglyoxal-H1 (MG-H1; 228 Da),and 3-deoxyglucosone-H (3DG-H; 318 Da). (d) Monolysyl glycation adducts: fructosyl-lysine (FL; 308 Da), N�-carboxymethyl-lysine (CML; 204 Da), and N�-carboxyethyl-lysine (CEL; 218 Da). (e) Cross-links and fluorophores: pentosidine (379 Da),(methylglyoxal-derived lysine dimer (MOLD; 341 Da), and argpyrimidine (254 Da). (f) Oxidation and nitration markers:methionine sulfoxide (MetSO; 165 Da) and 3-nitrotyrosine (3-NT; 226 Da). (c through f) Protein glycation, oxidation, and nitrationresidues are shown. For the corresponding free adducts at physiologic pH, the N-terminal amino group is protonated �NH3

� andthe C-terminal carbonyl is a carboxylate �CO2

� moiety.

1472 Journal of the American Society of Nephrology J Am Soc Nephrol 16: 1471–1485, 2005

and aliquots of dialysate from HD patients (three with diabetes) werecollected at the start and at the end of a 4-h dialysis session using apolysulfone membrane. Blood samples from PD patients (two withdiabetes) were collected 2 h after introduction of PD fluid into theperitoneal cavity during a peritoneal equilibration test (PET) withperitoneal dialysate collected after 0, 2, and 4 h of dwell time. Peritonealdialysate was also collected for approximately 12 h during the nightpreceding the test and for the 24 h before this. Peritoneal dialysate fromfour exchanges was pooled to estimate the 24-h excretion flux. The PDfluids used by patients in this study were single-compartment fluids(Dianeal; Baxter Healthcare Corporation, Deerfield, IL) that containedglucose osmolyte. Analyte estimates for PD and HD patients withdiabetes were not outliers from PD and HD patients without diabetes,respectively. Blood cells were sedimented by centrifugation (2000 � g,10 min), and the plasma was removed and immediately frozen at�80°C. Urine samples were collected at ambient temperature over 24 hfrom normal healthy control subjects, CRF patients and PD patientswith residual diuresis (�10% of analyte amounts were lost during thisperiod). Plasma and urine samples were stored at �80°C before anal-ysis. The study was approved by East London and The City HealthAuthority Research Ethics Committee (London, UK), and written in-formed consent was given by all participants and conformed to theDeclaration of Helsinki.

Other Clinical MarkersProtein glycation adducts were assayed by liquid chromatography

with triple quadrupole mass spectrometric detection (LC-MS/MS) (4).The interbatch coefficients of variation were �10%, and analyte esti-mates corroborated with independent measurements when these wereavailable (6,12). Free glycation adducts were determined by assay ofanalytes in ultrafiltrate (12 kD filter cutoff, 50-�l aliquot) of plasma,urine, and dialysate. Glycation adduct residues of plasma protein weredetermined in exhaustive enzymatic digests (50-�g protein equivalent)(4,13). Routine clinical service methods were used for urine and plasmacreatinine (Jaffe rate method) and serum albumin (BCG colorimetricassay) on an Olympus analyzer. Plasma monocyte chemotactic pro-tein-1 (MCP-1) and TGF-� were assayed in platelet free plasma bydouble-antibody ELISA (R & D Systems, Abingdon, UK).

Statistical AnalysesRenal clearance of analytes (ml/min) for control subjects and

CRF patients was determined as [Analyte]urine � urine volume/([Analyte]plasma� urine collection time). Clearance of analytes in PDpatients was calculated similarly using both urine and peritoneal dia-lysate outputs. Significance of difference between mean and medianAGE concentrations was determined using t test and the Mann Whit-ney U test, respectively. Correlation analysis was performed by calcu-lating Spearman � statistic. Mass transfer area coefficients (MTAC)were deduced by the simplified Garred equation (14).

ResultsDetection of Glycation, Oxidation, and Nitration FreeAdducts by LC-MS/MS in Plasma and Urine of ControlSubjects and CRF Patients and Plasma, Urine, andDialysate of ESRD Patients on HD or PD Therapy

Protein glycation, oxidation, and nitration free adducts weredetected by LC-MS/MS in plasma and urine of normal healthycontrol subjects, patients with CRF, and ESRD patients on PDand HD therapy. They were also detected in peritoneal dialy-sate and hemodialysate of ESRD patients (Figure 2, a throughh). Analytical chromatograms of the hydroimidazolone MG-H1showed the expected partially resolved pair of epimers (Figure2, c and d), and the hydroimidazolone 3DG-H showed theexpected three resolved structural isomers (4) (Figure 2, e andf). The concentrations of 3-NT were similar to those reported byothers using mass spectrometric techniques considered to beartifact-free (6).

Glycation, Oxidation, and Nitration Free Adducts in Plasmaand Urine of Control Subjects and CRF Patients

The AGE free adducts MG-H1 and 3DG-H were found in thehighest concentration in blood plasma of control subjects, 122nM, which was 133- and 46-fold higher than the concentrationsof pentosidine and 3-NT free adduct (Table 2). Most glycationfree adducts were increased in CRF patients, with respect tonormal control subjects. The increases were two-fold for FL,

Table 1. Clinical data for normal healthy control subjects and patients with CRF and ESRD on PD or HD therapya

Subjects n Age(Yr) M:F

PlasmaCreatinine

(�M)

CreatinineClearance(ml/min)

PlasmaAlbumin

(g/L)

Controls 8 45 � 6 6:2 98 � 16 107 � 22 46.4 � 1.1CRF 7 50 � 11 3:4 137 � 62 74 � 33

(P � 0.05)33.9 � 6.2(P � 0.001)

PD 8 52 � 16 3:5 639 � 130(P � 0.001; P� � 0.001)

ND 35.0 � 5.3(P � 0.001)

HD (before HD) 8 45 � 15 5:3 881 � 194(P � 0.001;

P� � 0.001; P� � 0.05)

ND 39.9 � 1.6(P � 0.001;

P� � 0.05; P� � 0.05)HD (after HD) 8 45 � 15 5:3 347 � 111

(P � 0.001; P� � 0.001;P� � 0.001; P� � 0.001)

ND 45.5 � 3.1(P� � 0.01;

P� � 0.001; P� � 0.001)aData are mean � SD. CRF, chronic renal failure; PD, peritoneal dialysis; HD, hemodialysis; ND, not determined. P, P�, P�,

and P� indicate significance with respect to control subjects and CRF, PD, and HD (before dialysis) patients, respectively.

J Am Soc Nephrol 16: 1471–1485, 2005 Glycation Free Adducts in Dialysis 1473

Figure 2. Specimen analytical chromatograms in the determination of protein glycation adducts by liquid chromatography withtriple quadrupole mass spectrometric detection in ESRD patients. CML (a) and [13C6]-CML (10 pmol; b) in plasma proteinhydrolysate of a hemodialysis (HD) patient, Rt 7.9 min. MG-H1 (c) and [15N2]-MG-H1 (50 pmol; d) in peritoneal dialysis (PD)dialysate at 2 h of dwell time, Rt 24.0 (MG-HA) and 24.2 (MG-HB) min. 3DG-H (e) and [15N2]-3DG-H (50 pmol; f), isomers 1, 2,and 3 (as indicated) in hemodialysate, Rts 24.6, 25.1, and 23.8 min, respectively. Argpyrimidine (g) and [15N2]argpyrimidine (50pmol; h) in HD patient plasma filtrate after a dialysis session, Rt 10.1 min. Chromatographic conditions were described in theMaterials and Methods section.


four-fold for CML, four-fold for CEL, four-fold for G-H1, five-fold for MG-H1, two-fold for 3DG-H, and two-fold for argpyri-midine. This was associated with a marked decline in renalclearance of glycation adducts but similar 24-h excretion rate,with respect to control subjects (Table 3). The plasma concen-tration of glycation and oxidation free adducts correlated neg-atively with creatinine clearance for CML (r �0.94, P � 0.01),MG-H1 (r �0.79, P � 0.05), and 3DG-H (r �0.75, P 0.05),consistent with a decrease in GFR leading to the accumulationof glycation free adducts in plasma. Glycation free adducts are

the major form of glycation adduct excretion in the urine ofhealthy subjects (4). The major glycation free adducts quanti-tatively excreted in the urine of control subjects and CRF pa-tients were FL, hydroimidazolones, CML, and CEL.

Glycation, Oxidation, and Nitration Free Adducts inPlasma, Urine, and Dialysate of PD Patients

In PD patients, the concentrations of most protein glycationfree adducts in blood plasma were increased, with respect tocontrol subjects. The increases were six-fold for CML, 10-fold

Table 2. Protein glycation, oxidation, and nitration free adduct concentrations in plasma of normal, healthy controlsubjects and patients with CRF and ESRD on PD or HD therapya

Study GroupProtein marker free adduct�Plasma (nM)

Control CRF PD HD (before Dialysis) HD (after Dialysis)

FL 69.7 � 4.3 121.9 � 20.6b 85.4 � 9.7 202.0 � 10.8d 125.7 � 11.3d

CML 18.9 � 3.1 66.4 � 12.8b 110.5 � 18d 220.9 � 10.9d 33.1 � 2.5b

CEL 35.1 � 5.7 126.7 � 24.1b 336.1 � 63.9d 740.0 � 74.7d 308.1 � 41.7d

G-H1 39.6 � 7.0 158.6 � 38.2b 148.9 � 22.9d 255.5 � 16.6d 139.3 � 12.9d

MG-H1 122 � 23 573 � 163b 2236 � 592d 4824 � 429d 1511 � 211d

3DG-H 122 � 15 208 � 15b 965 � 195d 1230 � 234d 341 � 42d

MOLD 2.97 � 0.68 2.34 � 0.59 2.56 � 0.63 9.45 � 0.99d 2.11 � 0.30Argpyrimidine 1.24 � 0.23 2.36 � 0.30c 1.90 � 0.23 5.50 (1.15 to 13.60) 2.23 � 0.59Pentosidine 0.92 � 0.46 1.13 � 0.52 5.07 � 0.37d 4.22 � 0.59d 1.30 � 0.27MetSO 19.9 � 3.06 22.8 � 3.63 18.6 � 1.3 31.0 � 5.3 27.0 � 4.13-NT 2.66 � 0.32 3.31 � 0.98 4.02 � 0.42c 3.74 � 0.56 2.97 � 0.44aData are mean � SEM. FL, fructosyl-lysine; CML, N�-carboxymethyl-lysine; CEL, N�-carboxyethyl-lysine; G-H1, glyoxal-

derived hydroimidazolone; MG-H1, methylglyoxal-derived hydroimidazolone; 3DG-H, 3-deoxyglucosone-derivedhydroimidazolone; MOLD, methylglyoxal-derived lysine dimer; MetSO, methionine sulfoxide; 3-NT, 3-nitrotyrosine.

bP � 0.01, cP � 0.05, and dP � 0.001 with respect to control subjects.

Table 3. Renal clearance of free glycation, oxidation, and nitration adducts of normal healthy control subjects andCRF patients, and urinary excretion rates of normal healthy control subjects, CRF patients, and ESRD patients onPD therapya

Subject GroupExcretion of Free Adduct (�mol/24 h) Clearance (ml/min)

Control CRF PD Control CRF PD

FL 5.87 � 0.61 3.46 � 0.19b 8.09 � 0.90c,e 64.5 � 6.0 10.6 � 4.4d 66.7 (37.8–222.2)CML 2.11 � 0.29 2.49 � 0.27 4.65 � 0.36d,g 73.5 � 6.3 28.0 � 3.5d 42.3 (14.0–75.4)b

CEL 3.06 � 0.41 5.08 � 0.83 6.47 � 0.66d 84.6 � 10.9 25.4 � 4.7d 9.2 (5.6–45.0)d

G-H1 3.03 � 0.37 2.77 � 0.45 4.09 � 0.70 48.0 � 6.1 14.9 � 3.3d 15.0 � 1.7b

MG-H1 6.62 � 0.68 10.76 � 2.13 58.52 � 6.21b,g 41.2 (22 to 121) 9.8 (7 to 38)b 16.8 � 3.8b

3DG-H 6.94 � 0.69 8.13 � 1.63 16.55 � 1.97d,e 38.1 (25 to 92) 26.2 � 6.7c 15.6 (3.9 to 33.5)b

Argpyrimidine 0.050 � 0.007 0.059 � 0.014 0.123 � 0.012d,e 31.4 � 6.0 21.3 � 6.2 6.4 � 2.3b

MOLD 0.039 � 0.005 0.029 � 0.004 0.053 � 0.0078 11.9 � 3.3 14.6 � 4.9 17.7 � 4.8Pentosidine 0.027 � 0.002 0.038 � 0.004c 0.082 � 0.002d,g 18.7 � 2.6 22.1 � 3.1 13.1 (4.4 to 26.9)MetSO 0.073 � 0.011 0.057 � 0.007 1.148 � 0.150d,g 2.4 � 0.3 1.9 � 0.2 41.7 � 5.5d

3-NT 0.013 � 0.002 0.023 � 0.004c 0.047 � 0.010b 12.5 � 2.3 7.3 � 2.5 4.9 (0.5 to 10.0)c

aData are mean � SEM except median (minimum–maximum) where the data are nonparametric. Excretion of free adductsfor control subjects and CRF patients was urinary and for PD patients was peritoneal dialysate and residual urinarycombined.

bP � 0.01, cP � 0.05, and dP � 0.001 with respect to control subjects.eP � 0.01, fP � 0.05, and gP � 0.001 with respect to CRF patients.


for CEL, four-fold for G-H1, 18-fold for MG-H1, eight-fold for3DG-H, five-fold for pentosidine, and 51% for 3-NT (Table 2).Protein glycation, oxidation, and nitration free adducts were

excreted by transfer into the peritoneal cavity and eliminationin the peritoneal dialysate and by residual diuresis. Althoughthe clearance of most protein glycation, oxidation, and nitration

Figure 3. Concentrations of protein glycation free adducts in peritoneal dialysate of PD patients showing the effect of PD fluid dwelltime. (a) FL. (b) CML. (c) CEL. (d) G-H1. (e) MG-H1. (f) 3DG-H. (g) pentosidine. (h) MOLD. Data are mean � SEM (n 8). The dottedline is the corresponding free adduct concentration in plasma at the 2-h dwell time in the peritoneal equilibration test (PET).


free adducts in PD patients was decreased with respect tocontrol subjects, the 24-h excretion rates were increased: G-H135%; FL, CML, CEL, argpyrimidine, and methylglyoxal-de-rived lysine dimer (MOLD) approximately two-fold; 3DG-Hand pentosidine three-fold; 3-NT four-fold; MG-H1 nine-fold;and MetSO 16-fold (Table 3). The proportion of protein glyca-tion, oxidation, and nitration free adduct excreted in the peri-toneal dialysate was 65% for FL, 76% for CML, 71% for CEL,78% for G-H1, 78% for MG-H1, 75% for 3DG-H, 90% for arg-pyrimidine, 74% for MOLD, 46% for pentosidine, 96% forMetSO, and 65% for 3-NT (two anuric patients were excludedfrom this calculation).

The concentration of most protein glycation, oxidation, andnitration free adducts in peritoneal dialysate increased withincreasing dwell time, except for MOLD and 3-NT, which didnot increase significantly (Figure 3 and Figure 4). From the 4-hPET, MTAC were deduced for 3DG-H and pentosidine freeadducts where peritoneal dialysate concentrations were consis-tent with equilibration with the plasma concentration. For theseand other analytes, dialysate/plasma concentration (D/P) ra-tios were deduced (Table 4). At the 4- and 12-h dwell times, theconcentrations of glycation free adducts FL, CML, CEL, G-H1,MG-H1, and MetSO in the peritoneal dialysate exceededgreatly the concentrations of the corresponding free adduct in

blood plasma determined at the 2-h dwell time point; hence, theD/P ratios were �1, and there were high peritoneal clearances(Table 4). The concentration of 3DG-H free adduct in peritonealdialysate at 12 h of dwell time exceeded the concentration of3DG-H free adduct in blood plasma. These data suggest intra-peritoneal formation of glycation and oxidation free adductswithin the PD fluid dwell time.

Glycation, Oxidation, and Nitration Free Adducts in Plasmaand Dialysate of HD Patients

In HD patients, the plasma concentrations of glycation freeadducts before a dialysis session were increased markedly,with respect to control subjects. The increases were three-foldfor FL, 12-fold for CML, 21-fold for CEL, seven-fold for G-H1,40-fold for MG-H1, 10-fold for 3DG-H, four-fold for argpyri-midine, three-fold for MOLD, and five-fold for pentosidine(Table 2). Similar high concentrations of glycation free adductswere found in the HD dialysate at the start of the dialysissession, except for MG-H1 and MOLD, reflecting rapid equili-bration of glycation free adducts across the dialysis membrane(Figure 5). Dialysis for 4 h normalized the plasma concentra-tions of pentosidine and MOLD free adducts and reversed theincreases of other glycation free adducts in blood plasma by 54to 93% but failed to normalize them (Table 2, Figure 5). The

Figure 4. Protein oxidative and nitrosative markers in plasma of normal healthy control subjects and chronic renal failure (CRF)patients and plasma and dialysate of PD patients. (A and B) Concentration of MetSO and 3-NT free adducts in peritoneal dialysatewith dependence on PD fluid dwell time. The dotted line is the corresponding free adduct concentration in plasma at the 2-h dwelltime in the PET. (C and D) Concentration of MetSO and 3-NT residues of plasma protein. CON, controls; PD (2), PD patients at2 h in the PET; HD (0) and HD (4), HD patients before and after a 4-h dialysis session, respectively. Data are mean � SEM (n 7 to 8). *P � 0.05, **P � 0.01, and ***P � 0.001, with respect to control subjects.


plasma concentrations of MetSO and 3-NT free adducts in HDpatients were increased but not significantly (Table 2).

The glycation free adduct reduction ratio (RR) was calculatedas the fall in concentration between the start and the end ofdialysis, expressed as a percentage of the concentration at thestart. Comparison was made with the RR for urea, which is anestablished measure of dialysis adequacy in a single sessionwith a minimum target of 65%. The urea RR was 69 � 8%. Freeadduct RR values were 84 � 5% for CML and 77 � 9% forMOLD (greater than urea, P � 0.001 and P � 0.05); 74 � 8% forMG-H1, 74 � 10% for 3DG-H, and 72 � 9% for pentosidine (allnot significant); and 39 � 19% for FL, 54 � 17% for CEL, 52 �

9% for G-H1 (lower than urea, P � 0.01, P � 0.05, and P �

0.001).

Glycation, Oxidation, and Nitration Adduct Residues inPlasma Proteins of Normal Control Subjects, CRF Patients,and ESRD Patients on HD or PD Therapy

The plasma protein content of FL residues in control subjectswas 0.83 mmol/mol lys. This was decreased to 0.34 mmol/mollys (�59%) in CRF patients and to 0.39 mmol/mol lys (�53%)in HD patients but was not decreased significantly in PD pa-tients (Figure 6a). FL residues degrade to form CML residues(15). The plasma protein content of CML residues was 0.039mmol/mol lys in control subjects and increased two-fold inCRF and PD patients and three-fold in HD patients. The plasmaprotein CML residue content of HD patients did not changeduring a dialysis session, but it was significantly higher than in

CRF and PD patients (Figure 6b). There was a lower plasmaprotein content of CEL residues than CML residues in controlsubjects (0.021 mmol/mol lys), and this was increased two-foldin all uremic patients (Figure 6c). The plasma protein content ofG-H1 residues was 0.049 mmol/mol arg in control subjects andwas increased significantly in HD patients only after a dialysissession (51%; Figure 6d). There was a high plasma proteincontent of MG-H1 residues in control subjects: 0.67 � 0.13mmol/mol arg. This was not increased in CRF patients but wasincreased approximately two-fold in PD and HD patients. Theplasma protein content of MG-H1 residues was also decreasedto control levels during a dialysis session in HD patients (Fig-ure 6e). The plasma protein content of 3DG-H residues incontrol subjects was lower than MG-H1 residues—0.37 � 0.04mmol/mol arg—and was increased two-fold in CRF and PDpatients and three-fold in HD patients before dialysis (Figure6f). The protein cross-links, pentosidine, and MOLD were mi-nor glycation adduct residues quantitatively in plasma proteinof control subjects (0.0104 � 0.0016 mmol/mol lys and 0.0087 �

0.0024 mmol/mol lys, respectively). The plasma protein con-tent of pentosidine residues was increased three-fold in CRFpatients, two-fold in PD patients, and four-fold in HD patientsbefore dialysis (Figure 6g). In contrast, there was no significantdifference in the plasma protein content of MOLD residues inuremic patients (Figure 6h). The plasma protein content ofMetSO residues of control subjects was 0.78 � 0.05 mmol/molmet and was increased approximately two-fold in CRF patients,PD patients, and HD patients before HD. The plasma proteincontent of MetSO residues was not increased significantly afteran HD session (Figure 4c). The plasma protein content of 3-NTresidues of control subjects was 0.0031 � 0.0009 mmol/mol tyrand was increased three-fold in PD patients and HD patientsbefore HD. The plasma protein content of 3-NT residues wasdecreased during HD (Figure 4d).

Plasma Concentrations of MCP-1 and TGF-�The plasma concentration of the chemokine MCP-1 was in-

creased significantly in PD and HD patients, with respect tocontrol subjects (Figure 7A); estimates in HD patients afterdialysis were corrected for loss of plasma water by normalizingto albumin concentration predialysis. The plasma concentrationof soluble TGF-� was increased in HD patients only, before andafter a dialysis session, with respect to control subjects and CRFand PD patients (Figure 7B).

Correlation Analysis of Protein Glycation, Oxidation, andNitration Adducts

The relationships between glycation, oxidation, and nitrationadduct residues and free adducts in normal control subjectsand CRF, PD, and HD patients were examined. For HD pa-tients, analyte values were predialysis estimates. Homotypiccorrelations are correlations of glycation, oxidation, and nitra-tion adducts of the same type in different forms or locations,namely plasma protein residues and free adducts in plasmaand urine. There were no homotypic correlations in controlsubjects and CRF patients. In HD and PD patients, the plasmaconcentration of CML free adduct correlated positively with the

Table 4. Dialysate/plasma concentration ratios andmass transfer area coefficients for the PETa

Solute D/P Ratio MTAC(ml/min)

Creatinine 0.69 � 0.08 9.3 � 2.9FL 7.8 � 4.2 —CML 3.6 � 2.4 —CEL 1.6 � 1.0 —G-H1 2.3 � 1.2 —MG-H1 1.5 � 0.7 —3DG-H 1.2 � 1.0 6.5 � 4.1Argpyrimidine 8.9 � 6.1 —Pentosidine 1.1 � 0.4 31.5 � 8.5MetSO 4.0 � 1.2 —MOLD 0.99 � 0.97 —3-NT 0.75 � 0.18 —

aData are mean � SD or median (minimum–maximum).PET, peritoneal equilibration test; D/P, dialysate/plasmaconcentration; MTAC, mass transfer area coefficients. TheD/P ratio is solute�Dialysate, 4 h/solute�Plasma, 2 h. SimplifiedGarred equation: MTAC (VPD/t) ln {VPDF � (Freeadduct�Plasma � Free adduct�PD, t 0)�/VPD � (Freeadduct�Plasma � Free adduct�PD, t 240)�} where VPDF andVPD are PD fluid volumes infused and peritoneal dialysatecollected, and Free adduct�Plasma, Free adduct�PD, t 0, andFree adduct�PD, t 240 are the glycation free adductconcentrations in the plasma and peritoneal dialysate at thestart and the end of the 4 h PET (14).


Figure 5. Concentrations of protein glycation free adducts in plasma and hemodialysate of HD patients. (a) FL. (b) CML. (c) CEL.(d) G-H1. (e) MG-H1. (f) 3DG-H. (g) Pentosidine. (h) MOLD. Key: PCON, control plasma ultrafiltrate; PHD (0), plasma ultrafiltrateof HD patients at the start of dialysis; PHD (4), plasma ultrafiltrate of HD patients after 4 h of dialysis; DL (0), dialysate at the startof dialysis; DL (4), dialysate after a 4-h dialysis session. Data are mean � SEM (n 8). **P � 0.01 and ***P � 0.001, plasmaconcentrations with respect to control subjects. oP � 0.05, ooP � 0.01, and oooP � 0.001 for plasma and dialysate at the end of adialysis session with respect to the corresponding concentration at the start of the dialysis session; �P � 0.05 and ��P � 0.001for dialysate at the start of a dialysis session with respect to plasma at the start of dialysis.


Figure 6. Concentrations of glycation adduct residues in plasma protein of normal control subjects, CRF patients, and ESRDpatients with PD and HD therapy. (a) FL. (b) CML. (c) CEL. (d) G-H1. (e) MG-H1. (f) 3DG-H. (g) Pentosidine. (h) MOLD. CON,controls; PD (2), PD patients 2 h after change of PD fluid; HD (0) and HD (4), HD patients before and after a 4-h dialysis session,respectively. Data are mean � SEM (n 7 to 8). *P � 0.05, **P � 0.01, and ***P � 0.001, with respect to control subjects; oP � 0.05with respect to CRF and PD patients; �P � 0.05 with respect to HD (0).


concentration of CML residues (r 0.51, P � 0.05), and theplasma concentration of 3DG-H free adduct correlated posi-tively with the concentration of 3DG-H residues (r 0.53, P �

0.05). Other correlation analyses of protein glycation, oxidation,and nitration adduct analytes and related variables are givenfor control subjects (Tables 5 through 7), CRF patients (Tables 8through 10), and PD and HD patients (Tables 11 and 12). Theseare provided, without detailed explanation, for substantiationand interpretation in future studies.

DiscussionIn this study, we report the marked accumulation of protein

glycation free adducts in CRF and ESRD. Glycation free ad-ducts normally represent �5% of the total glycation adducts inplasma, but this increases substantially for some glycation ad-ducts in uremia—particularly MG-H1. Overall, the total con-centration of glycation free adducts in blood plasma increasedin the order control�CRF�PD�HD (predialysis) subjects. Thispattern of high plasma concentrations of glycation free adductsin uremic patients reflects marked decreased clearance of gly-cation free adducts in CRF patients. Marked decreased clear-ance was also generally found in PD patients, in whom theeffect was exacerbated by increased glycation free adduct for-

mation—as indicated by increased glycation free adduct excre-tion. The increased plasma concentration of glycation free ad-ducts in HD may also be due to increased formation anddecreased clearance of glycation free adducts.

In CRF patients, compared with creatinine, we found a dis-proportionate increase in plasma concentration of glycationfree adducts and precipitous decline in their clearance whileexcretion rates were normal—except for FL and pentosidine.This may indicate that there is active tubular secretion of free

Table 6. Correlation of protein glycation, oxidation, andnitration free adducts in plasma in normal, healthycontrol subjects

Analyte CML CEL G-H1 MG-H1 3DG-H

CML 0.79a 0.89b

CEL 0.71a

G-H1 0.79a 0.73a

MG-H1 0.71a

3DG-H 0.89b 0.73a

aP � 0.05. bP � 0.01.

Figure 7. Plasma concentrations of monocyte chemotactic protein-1 (MCP-1) and TGF-� of normal control subjects, CRF patients,and ESRD patients with PD and HD therapy. (a) MCP-1. (b) TGF-�. CON, controls; PD (2), PD patients 2 h after change of PD fluid;HD (0) and HD (4), HD patients before and after a 4-h dialysis session, respectively. Data are mean � SEM (n 7 to 8). *P � 0.05and **P � 0.01 with respect to control subjects. A correction factor using the individual changes in serum albumin concentrationsbefore and after dialysis was applied to post-HD (4) data to allow for plasma water loss during HD (mean correction factor 0.88).

Table 5. Correlation of protein glycation, oxidation, and nitration adduct residues in plasma protein in normal,healthy control subjects

Analyte FL CEL G-H1 3DG-H Pentosidine MetSO

FL �0.86a �0.71b

CEL �0.86a

G-H1 0.91a

3DG-H 0.71b

Pentosidine �0.71b 0.91a

MetSO 0.71b

aP � 0.01. bP � 0.05.


adducts and this is impaired in CRF. Some other uremic toxinswere actively secreted in renal tubules by organic anion trans-porters (16). The plasma concentration of glycation free adductscorrelated negatively with creatinine clearance, but there wasno correlation of glycation free adduct clearances with molec-ular mass (data not shown). This is probably because glycationfree adducts all have low molecular mass (�400 Da) and henceare freely filtered by the glomerulus.

In PD therapy, excretion of protein glycation, oxidation, andnitration free adducts occurs mainly by elimination in the peri-toneal dialysate—except for pentosidine. D/P ratios were �1for most analytes (FL, CML, CEL, G-H1, MG-H1, argpyrimi-dine, and MetSO) at the end of the 4-h PET. Glycation andoxidation free adducts are proposed to pass through the hy-pothesized small 5-nm pores of micromolecular transport (17),as indeed do amino acids (18), although additional active trans-port may enhance solute entry into the peritoneal cavity; cf therole of aquaporin-1 in the transport of water (19). The probable

explanations for glycation and oxidation free adduct concen-trations in the peritoneal dialysate being higher than in plasmaare (1) formation of glycation and oxidation free adducts in theperitoneal cavity by glycation and oxidation of amino acids andmatrix proteins (with subsequent proteolysis) by glucose deg-radation products in the PD fluid and (2) active transport ofglycation and oxidation free adducts across the endothelium ofperitoneal capillaries and vessels. Increased FL (as indicated bythe assay of furosine), 3DG-H, other AGE, and oxidative mark-ers in protein of peritoneal dialysate, vascular wall, and me-sothelium have been reported (20–24) and were decreased byuse of low glucose degradation products that contained PDfluid (24). Glycation and oxidation free adducts probably utilizeamino acid transporters to cross the capillary endothelium of

Table 10. Correlation of protein glycation, oxidation,and nitration free adduct urinary excretion rates inCRF patients

Analyte CEL G-H1 3DG-H Pentosidine MetSO

CEL 0.86a

G-H1 0.94b

3DG-H 0.86a 0.94b

Pentosidine 0.89a

MetSO 0.89a

aP � 0.05. bP � 0.01.

Table 7. Correlation of protein glycation, oxidation, and nitration free adduct urinary excretion rates in plasma innormal, healthy control subjects

Analyte CML CEL MG-H1 3DG-H MOLD MetSO

CML 0.71a 0.76a

CEL �0.82a

MG-H1 0.93b 0.86a 0.79a

3DG-H 0.71a �0.82a 0.93b

MOLD 0.86a 0.93c

MetSO 0.76a 0.79a 0.93c

aP � 0.05. bP � 0.01. cP � 0.001.

Table 8. Correlation of protein glycation, oxidation, and nitration adduct residues in plasma protein in CRFpatients

Analyte FL CEL MG-H1 3DG-H Pentosidine MetSO 3-NT TGF-�

FL 0.81a

CEL 0.81a

MG-H1 0.94b 0.89b

3DG-H 0.94b 0.94b

Pentosidine 0.86a 0.82a

MetSO 0.86a

3-NT 0.82a

TGF-� 0.89b 0.94b

aP � 0.05. bP � 0.01.

Table 9. Correlation of protein glycation, oxidation, andnitration free adducts in plasma in CRF patients

Analyte FL 3DG-H

FL �0.82a

3DG-H �0.82a

aP � 0.05.


the peritoneum (25). This transport may contribute to aminoacid loss into the peritoneal dialysate in PD therapy (18). TheMTAC value for creatinine was similar but pentosidine washigher than reported values (20); the MTAC for 3DG-H waslower than that of creatinine.

The excretion of most protein glycation, oxidation, and nitra-tion free adducts was increased in PD patients, with excretionsof MetSO and MG-H1 exceptionally high. This may reflectincreased protein modification in carbonyl, oxidative, and ni-trosative stress of uremia. With reduced functional kidney massand elimination via the peritoneal cavity, MetSO escapes me-tabolism by renal MetSO reductase (26); hence, the excretionrate and clearance of MetSO were increased markedly (16-fold).The nine-fold increase in excretion of MG-H1 in PD patientswas the most marked increase found for the excretion of aglycation free adduct. Increased protein glycation by MG inESRD is expected because of the high concentrations of MGfound in the plasma of ESRD patients and also in PD fluids(9–11,27).

HD therapy of ESRD patients removed glycation free ad-ducts from the circulation by passage through the dialysismembrane. Protein glycation and oxidation free adducts havemolecular masses �400 Da and therefore are small molecular

mass uremic toxins (2). At the end of the dialysis session,protein glycation free adducts were decreased equally in bothplasma and hemodialysate but not normalized to control lev-els—except for pentosidine and MOLD. RR values of glycationfree adducts ranged from 39 to 84%, demonstrating heteroge-neity of clearance, and were lower than urea for only FL, CEL,and G-H1. Increased frequency of HD with higher flux mayimprove the elimination of glycation free adducts.

Most protein glycation, oxidation, and nitration adduct moi-eties in plasma are modified amino acid residues of plasmaprotein and are not eliminated efficiently by PD and HD ther-apy. The glycation adduct residue content of plasma proteinmay be affected by hypoalbuminemia in renal failure and re-lated changes in albumin turnover (Table 1). Hypoalbumine-mia in PD patients is caused by loss of albumin in urine andperitoneal dialysate and is countered by increased albuminsynthesis—inflammation and poor nutrition limit this response(28). This leads to a decreased half-life of albumin. Glycation ofproteins in CRF and ESRD is expected to increase because ofincreased concentrations of �-oxoaldehyde glycating agents(9,10,27). The concentration of FL residues may be decreased inCRF and HD patients by increased oxidative stress with con-version of FL to CML residues (15). High levels of glycoxidation

Table 11. Correlation of protein glycation, oxidation, and nitration adduct residues in plasma protein in HD(predialysis) and PD patients

Analyte Albumin FL CML CEL MG-H1 3DG-H MetSO 3-NT

Albumin �0.56a

FL 0.57a �0.53a

CML 0.57a 0.63b

CEL 0.63b �0.53a

MG-H1 �0.56a

3DG-H �0.53a 0.66b 0.58a

MetSO �0.53a 0.66b

3-NT 0.58a

aP � 0.05. bP � 0.01.

Table 12. Correlation of protein glycation, oxidation, and nitration free adducts in plasma protein in HD(predialysis) and PD patients

Analyte FL CML CEL G-H1 MG-H1 3DG-H Argpyrimidine Pentosidine MetSO 3-NT TGF-�

FL 0.90a 0.76a 0.75b 0.64c �0.67b

CML 0.90a 0.87a 0.82a 0.76a 0.59c �0.51c

CEL 0.76a 0.87a 0.75a 0.67b 0.62c

G-H1 0.75c 0.82a 0.75a 0.72b �0.51c �0.51c

MG-H1 0.64c 0.76a 0.67b 0.72b 0.68b 0.68c �0.59c

3DG-H 0.68b �0.83c

Argpyrimidine 0.68c

Pentosidine �0.51c

MetSO 0.59c 0.62c

3-NT �0.67b �0.51c �0.51c �0.59c

TGF-� �0.83aP � 0.001. bP � 0.01. cP � 0.05.


adducts (pentosidine and CML) in HD may reflect increasedoxidative stress, relative to PD (29). The concentrations of MG-H1, MetSO, and 3-NT residues were normalized in HD patientsafter dialysis. 3-NT–modified albumin undergoes preferentialendothelial transcytosis (30), which may be increased in theinflammatory response to dialysis. MetSO residues may bereduced to methionine residues by MetSO reductase duringendothelial transcytosis. MG-H1–modified proteins may bebound by activated monocytes in the dialysis session and de-graded (31). The concentration of MG-H1 residues in plasmaprotein of PD and HD patients correlated negatively with theplasma concentration of albumin (Table 11). Decreased albuminconcentration is associated with poor survival of ESRD patients(23).

The few homotypic correlations of glycation adduct proteinresidues and free adducts reflect important nonvascularsources of glycation free adduct formation: Degradation ofglycated tissue proteins (4), adsorption from digested foodprotein (8), and glycation of amino acids (13). Major sources ofglycation and oxidation free adducts are likely to be cellularproteolysis and digested food (4,32).

The plasma concentration of MCP-1 was increased signifi-cantly in PD and HD patients, and the plasma concentration ofsoluble TGF-� was increased in HD patients only, confirmingprevious reports (33–36). MCP-1 is associated with macrophageinfiltration into the peritoneal cavity of PD patients and corre-lates with soluble adhesion molecule expression in HD (33,37).TGF-� is a key mediator of renal fibrosis in progressive renalfailure and ESRD. The concentration of MG-H1 and 3DG-Hresidues in plasma protein correlated positively with TGF-� inCRF patients. Increased expression of TGF-� in CRF is associ-ated with a progressive decline of residual renal function andincreased fibrosis leading to ESRD. Although protein glycation,oxidation, and nitration free adducts are eliminated by dialysis,plasma MCP-1 and TGF-� concentrations were maintained bydialysis therapy. The inflammation of dialysis and the increaseAGE may be linked, but there is no direct relationship with theclearance of AGE free adducts.

AcknowledgmentsWe thank Baxter Healthcare Inc (Deerfield, IL) for support for a PhD

studentship for S.A. and the Wellcome Trust for support for our proteinbiomarker research. We thank the staff and patients of the Renal Unit,St. Bartholomew’s Hospital, for their participation in this study.

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