Identification of -dicarbonyl scavengers for

cellular protection against carbonyl stress

Georg T. Wondrak, Daniel Cervantes-Laurean, Michael J. Roberts, Jaber G. Qasem, Moonsun Kim, Ela

Department of Pharmacology and Toxicology, College of Pharmacy, Arizona Cancer Center, University of Ariz
Received 9 March 2001;
accepted 26 July 2001.
Available online 28 December 2001.

Abstract
Tissue deterioration and aging have long been associated with the accumulation of chemically
induced protein and DNA damage. Reactive oxygen species (ROS) and reactive carbonyl species
(RCS), especially -dicarbonyl compounds, are key mediators of damage caused by oxidative
stress, glycation, and UV-irradiation. The toxic effects of ROS are counteracted in vivo by
antioxidants and antioxidant enzymes, and the deleterious effects of one RCS, methylglyoxal, are
counteracted by a ubiquitous glyoxalase system. Carbonyl stress as a result of toxic effects of
various mono-dicarbonyls (e.g. 4-hydroxynonenal) and -dicarbonyls (e.g. glyoxal and
deoxyosones) cannot be directly antagonized by antioxidants, and only a small number of
biological carbonyl scavengers like glutathione (GSH) have been identified to date. We have
developed a new screening method for the identification of carbonyl scavengers using a rapid
glycation system that proceeds independent of oxygen and therefore, excludes identification of
inhibitory compounds acting as antioxidants. Using this screening assay adapted to 96-well
microtiter plates, we have identified the cysteine derivative 3,3-dimethyl- -cysteine as a potent
inhibitor of non-oxidative advanced glycation. Comparative kinetic analyses demonstrated the
superior -oxoaldehyde-scavenging activity of -penicillamine over that of aminoguanidine. Penicillamine traps -oxoaldehydes by forming a 2-acylthiazolidine derivative as shown by
structure elucidation of reaction products between -penicillamine and methylglyoxal or
phenylglyoxal. We demonstrated that upon co-incubation, -penicillamine protects human skin
keratinocytes and fibroblasts (CF3 cells) against glyoxal- and methylglyoxal-induced carbonyl

This Article

Abstract

toxicity. Our research qualifies -amino--mercapto-,-dimethyl-ethane as a promising

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pharmacophore for the development of related -dicarbonyl scavengers as therapeutic agents to

protect cells against carbonyl stress.
Services

Author Keywords: Glycation; -Dicarbonyl compounds; Carbonyl scavenger; HaCat

keratinocytes; CF3 fibroblasts; -Penicillamine
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Abbreviations: ADP-ribose, adenosine 5-diphosphoribose; AGE, advanced glycation end

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product; AGEBSA, BSA modified with advanced glycation end products; CML, N Alert me to new issues of the journal

carboxymethyllysine; DTPA, diethylenetriamine-pentaacetic acid; GOLD, glyoxallysine dimer;

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managerlaser desorption ionization-time of flight-mass spectrometry;
MALDI-TOF-MS,
matrix-assisted

MOLD, methylglyoxallysine dimer; NAC, N-acetyl- -cysteine; RCS, reactive carbonyl

species; ROS, reactive oxygen species.
2009 International Society for Peritoneal Dialysis
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Part 2: Cellular and Molecular Biology of the Peritoneum and Peritoneal Dialysis

QUANTITATION OF MARKERS OF PROTEIN DAMAGE BY GLYCATION,

OXIDATION, AND NITRATION IN PERITONEAL DIALYSIS
Naila Rabbani and Paul J. Thornalley

Warwick Medical School, Clinical Sciences Research Institute, University of Warwick,

University Hospital, Coventry, U.K.
Correspondence to: P.J. Thornalley, Clinical Sciences Research Institute, Warwick
Medical School, University of Warwick, University Hospital, Clifford Bridge Road,
Coventry CV2 2DX, U.K. P.J.Thornalley@warwick.ac.uk
TOP
ABSTRACT
MEASUREMENT OF GLYCATED,...
MEASUREMENT OF GLYCATED AND...
PROTEIN CARBONYLS AND ADVANCED...
SKIN AUTOFLUORESCENCE
PROFOUND MISHANDLING OF...
EXCRETION RATES OF GLYCATION,...
CONCLUSIONS
REFERENCES

ABSTRACT
Proteolysis products of proteins damaged by glycation, oxidation, and nitration
glycated, oxidized, and nitrated amino acids (glycation, oxidation, and nitration
free adducts)are waste products normally excreted in urine and cleared in
peritoneal dialysate. Glucose degradation products in peritoneal dialysis (PD)
fluids may increase protein damage, giving rise to increased protein glycation,
oxidation, and nitration adduct residues of proteins and increased flux of
glycation, oxidation, and nitration free adducts. Increased protein damage has
been linked to mortality in end-stage renal disease. Reliable quantitation of
markers for adducts of protein glycation, oxidation, and nitration is required for

mechanistic studies and for morbidity and mortality risk analysis in PD patients.
We review the available analytical techniques for such quantitation. Stable
isotopic dilution analysis with tandem mass spectrometry is the "gold standard."
This method needs to be applied further in the study of PD and to validate other
techniques so that the effect of PD on the metabolism and clearance of damaged
proteins and related products can be quantified, and so that best-practice fluid
management can be established to minimize cardiovascular risk.
KEY WORDS: Glycation; oxidative stress; nitrosative stress; tandem mass
spectrometry; immunoassay; skin autofluorescence.
Peritoneal dialysis (PD) is a method of renal replacement therapy of arguably preferred
first use in renal failure. It is associated with better preservation of residual renal function
than that seen in hemodialysis, and with related benefits (1). However, an adverse
feature of conventional dialysis fluids with high concentrations of glucose osmolyte has
been the presence of glucose degradation products (GDPs), which are potentially
damaging. During heat sterilization of PD fluids, GDPs are formed from the oxidation,
dehydration, and fragmentation of glucose.
Dicarbonyls, which are also trace metabolites, constitute a major class of GDPs.
Important dicarbonyl GDPs are glyoxal, methylglyoxal, 3-deoxyglucosone (3-DG), and
3,4-dideoxyglucosone-3-ene (2,3). Glucose and GDPs are important glycating agents in
the peritoneum and elsewhere, and the peroxide produced from the autoxidation of
glucose and some buffer components contributes to oxidative and nitrosative damage to
proteins. Protein damage is linked to loss of protein structure and function, decreased
mesothelial cell viability (4), and increased detachment of endothelial cells from the
vascular extracellular matrix (5)all linked to breakdown of the ultrafiltration properties
of the peritoneal membrane (6) and increased risk of cardiovascular disease (7). Best
efforts should therefore be made to decrease protein damage in PD therapy, while
preserving ultrafiltration and flow of uremic toxins into the peritoneal cavity for removal in
dialysate.
Proteins damaged by glycation, oxidation, and nitration contain glycation, oxidation, and
nitration adduct residues. The initial glycation adducts formed during glycation by
glucose and other monosaccharides are called early-stage glycation adducts. Glucose
reacts with lysine residue side-chain amino groups to form, initially, a Schiff base adduct
that slowly rearranges to N -fructoselysine (FL). Schiff base and FL adduct residues are
early glycation adducts. Schiff base and FL adducts slowly degrade in further advanced
reactions to form many different glycation adducts. These adducts are collectively called
"advanced glycation end-products" (AGEs). Dicarbonyls react directly with proteins, also
forming AGEs.

Quantitatively important AGEs are hydro-imidazolones derived from arginine residues

modified by glyoxal, methylglyoxal, and 3-DG: N -(5-hydro-4-imidazolon-2-yl)ornithine,
N -(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine (MG-H1), N -(5-hydro-5-(2,3,4trihydroxybutyl)-4-imidazolon-2-yl)ornithine, and related structural isomers (3DG-H).
Other important and widely studied AGEs are N -carboxymethyllysine (CML) and N carboxyethyllysine (CEL) and the protein crosslinks pentosidine and glucosepane.
Further AGEs and related derivatives of emerging importance are N carboxymethylcysteine, N -carboxymethylarginine and ornithine (the latter formed as a
degradation product of hydro-imidazolones) (8). Important markers of protein oxidation
are methionine sulfoxide (MetSO) residues (formed by exposure to hydrogen peroxide,
hypochlorite, and peroxynitrite), dityrosine [formed by exposure of two proximate
tyrosine residues in proteins to hydrogen peroxide (including peroxidase-catalyzed
reactions), hypochlorite, and peroxynitrite (the efficacy of formation depending on the
side chain moieties for crosslink formation)]; and N-formylkynurenine [NFK (formed by
exposure of tryptophan residues in proteins to hydrogen peroxide, hypochlorite, and
peroxynitrite under physiologic conditions)] (9). Protein nitration leads to the formation of
3-nitrotyrosine (3-NT) residues by the interaction of proteins with peroxynitrite and nitryl
chloride (10).
The extent of these types of protein modification is usually 0.01% 5%. Proteins
damaged in this manner undergo cellular proteolysis and release glycated, oxidized,
and nitrated amino acids called glycation, oxidation, and nitration free adducts.
Glycation, oxidation, and nitration free adducts are the major forms in which proteins are
usually excreted from the body in urine (11). They are also the major forms in which
damaged or modified protein are cleared from the body in dialysate during PD (12).
Measurement of the amounts of glycation, oxidation, and nitration free adducts in PD
dialysate pooled over a 24-hour period, combined with their excretion in residual
diuresis, provides an estimate of the flux of protein damage in a PD patient. (Glycation,
oxidation, and nitration free adducts absorbed from digested proteins in foods contribute
to the measured quantity, but this contribution may be minor in PD patients because of
the increased endogenous formation of damaged proteins.)

MEASUREMENT OF GLYCATED, OXIDIZED, AND

NITRATED PROTEINS AND RELATED FREE
ADDUCTS BY STABLE ISOTOPIC DILUTION

ANALYSIS
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PROTEIN CARBONYLS AND ADVANCED...
SKIN AUTOFLUORESCENCE
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CONCLUSIONS
REFERENCES

Measurement of damage involves detection of the glycated, oxidized, and nitrated

amino acids in the presence of an excess (by a factor of about 100 to 1 million) of
unmodified amino acids and many other potential interfering substances. The best
technique available to meet this analytical challenge is stable isotopic dilution analysis
using liquid chromatography with positive-ion electrospray-ionization tandem mass
spectrometric detection (LC-MS/MS). Protein substrates are hydrolyzed, and the
glycation, oxidation, and nitration adducts are thereafter quantified. Enzymatic
hydrolysis of protein substrates is used, because several analytes are labile in
conventional acid or base chemical hydrolysis of proteins.
The exhaustive enzymatic hydrolysis of protein substrates involves a cocktail of
enzymes: pepsin, pronase E, prolidase, and aminopeptidase. Steps must be taken to
avoid oxidative degradation of the adduct residues of proteins during enzymatic
hydrolysis; the addition of the antioxidant thymol and incubation under nitrogen or argon
are typical methods (11,13). After an initial step with pepsin (replaced by collagenase for
analysis of collagen) under acidic conditions (14), antibiotics are included in the
enzymatic digest to prevent bacterial growth in the amino acid solution being produced
(13). These procedures yield acceptable analyte stabilities and exhaustive proteolysis,
and then proceed to near-completion for proteins modified only minimally by glycation,
oxidation, and nitration. Correction of the analytes detected in enzymatic hydrolysates is
made for glycated, oxidized, and nitrated amino acids released by autohydrolysis of the
proteolytic enzymes added.
Recently, we automated this process with a simple robotic system. For the LC-MS/MS,
we used a graphitic stationary phase such as the Hypercarb column (Thermo Hypersil,
Bellefonte, PA, U.S.A.) with column switching. This technique facilitates the retention
and sequential elution of analytes of diverse hydrophobicity and column washing (11).

The method was reported initially for FL and for 12 AGEs, 2 oxidation markers, and the
nitration marker 3-NT. Further analytes have since been added (Table 1) and
chromatographic procedures have been further customized to complete data collection
of all analytes in a 35-minute run.

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table:
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window]
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TABLE 1 Markers of Protein Glycation, Oxidation, and Nitration

Determined by Stable Isotopic Dilution Analysisa

Analysis of exhaustive digests of protein gives the amounts of protein glycation,

oxidation, and nitration free adduct residues normalized to the amounts of protein
(picomoles analyte per milligram protein) or to the corresponding unmodified amino acid
(millimoles analyte per mole unmodified amino acid). Analysis of analytes in ultrafiltrates
(12 kDa cut-off membrane filter) of plasma, urine, or dialysate yields the concentrations
of glycation, oxidation, and nitration free adducts to glycated, oxidized, and nitrated
amino acids. The release of further free adducts by enzymatic digestion of the
ultrafiltrate gives the amount of protein glycation, oxidation, and nitration adduct residues
in low molecular mass polypeptides that are called "glycated, oxidized, and nitrated
peptides."
Stable isotopic dilution analysis with LC-MS/MS detection is the "gold standard"
reference technique for analysis of protein glycation, oxidation, and nitration free
adducts. The major restriction to the technique is the availability of isotope-substituted
standards for a comprehensive range of analytes, given that most are not available
commercially. The overwhelming advantage of the technique is that, from a small
sample (25 g protein and 25 L ultrafiltrate), it can provide a relatively comprehensive
and quantitative analysis of protein glycation, oxidation, and nitration adduct residues
and free adducts. In future research it will be important to use LC-MS/MS in studies of
protein damage in PD and also to corroborate other methods for quantifying adducts of
protein glycation, oxidation, and nitration to the LC-MS/MS reference method.

MEASUREMENT OF GLYCATED AND NITRATED

PROTEINS AND RELATED FREE ADDUCTS BY
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ABSTRACT
IMMUNOASSAY
MEASUREMENT OF GLYCATED,...
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CONCLUSIONS
REFERENCES

Protein glycation and nitration adduct residues are often detected by immunoassay. In
quantifying such adducts by immunoassay, doubts arise over the reliability of the data
obtained. Several problems have been identified with immunoassay procedures for
markers of protein damage:

Incomplete characterization of antibody specificity

Varying affinities of antibodies for marker adduct residues of proteins and free adducts

Presence in the assay protocol of adduct residues of protein damage in proteins used to
block nonspecific antibody binding (for example, in dried milk protein)

Use of highly modified standard antigens dissimilar to the minimally modified antigens in
physiologic samples

For these reasons, immunoassay often does not provide absolute concentrations or
amounts of analyte, but rather arbitrary units with or without normalization to a reference
glycated, oxidized, or nitrated protein standard. Major disparities in immunoassay
detection of analytes of protein damage have been observed: the monoclonal antibody
6D12 used to detect CML residues was later found to detect CEL also (15,16), and
detection of 3-NT residues in plasma protein by immunoassay and LC-MS/MS showed
discrepancies with factors of 50 100 (17).

PROTEIN CARBONYLS AND ADVANCED

PROTEIN OXIDATION PRODUCTS AS MARKERS
OF PROTEIN OXIDATION
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REFERENCES

"Protein carbonyls" have been used as a measure of protein oxidation. Total protein
carbonyls have been determined by derivatization with 2,4-dinitrophenylhydrazine, with
the related hydrazones formed being detected by characteristic absorbance at 360
390 nm, by immunodetection, or by reduction with tritiated borohydride. Protein
carbonyls are considered to be mainly 2-aminoadipic semi-aldehyde (AASA) formed
from oxidative deamination of lysine, and glutamic semi-aldehyde formed by oxidation of
proline and arginine residues. The AASA may oxidize further to 2-aminoadipic acid,
which has been detected in human skin collagen (18). These analytes have been
detected discretely after reduction to 6-hydroxy-2-aminocaproic acid and 5-hydroxy-2aminovaleric acid (19). There is doubt concerning whether measurement of protein
carbonyls is reporting oxidative damage; in a recent validation, this putative measure of
protein oxidation was unresponsive in models of oxidative stress (20).
"Advanced oxidation protein products" (AOPPs) are a further indirect measure of protein
oxidation in which molecular species contributing to the assessment have been
incompletely defined. This measure of the ability of protein oxidation products to oxidize
iodide to iodine is thought to be related to N-chloramine derivatives formed by oxidation
of protein with hypochlorite generated by myeloperoxidase. It is unclear whether AOPPs
have significance for oxidative damage of proteins. Some studies have claimed that
AOPPs increase with progression of renal failure (21); others have found that AOPPs
decrease after initiation of PD therapy (22).

SKIN AUTOFLUORESCENCE
TOP
ABSTRACT
MEASUREMENT OF GLYCATED,...
MEASUREMENT OF GLYCATED AND...
PROTEIN CARBONYLS AND ADVANCED...
SKIN AUTOFLUORESCENCE
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CONCLUSIONS
REFERENCES

Recent advances in instrumentation have seen the development a bedside fluorometer

(autofluorescence reader). The instrument illuminates 1 cm2 of skin with an excitation
wavelength band of 300 420 nm (peak excitation: 350 nm). Emitted light from the skin
is measured over the wavelength range 300 600 nm. Autofluorescence is calculated
by dividing the average light intensity emitted per nanometer over the 420 600 nm
range by the average light intensity emitted per nanometer over the 300 420 nm
excitation range. Autofluorescence of the skin (SAF) is measured 6 times over a 50second period (every 10 seconds) at the volar side of the arm about 10 cm below the
elbow fold and at the dorsal side of the lower leg (calf). In hemodialysis patients, SAF
correlated with skin biopsy content of pentosidine (r = 0.75) and with CML and CEL
(both r = 0.45). Hence, measurements of SAF have been interpreted as a measurement
of AGEs. There are problems with this interpretation:

Most AGEs are not fluorescent, particularly the quantitatively important hydroimidazolone AGEs and CML and CEL.

The fluorescence characteristics used in this assay are not specific for fluorescent AGEs.

Fluorescence in proteins is a result of multiple fluorophores that compete for the same
excitation energy in the detection, and hence, there is no direct relationship between
concentration of the adduct and fluorescence.

The main components of SAF spectra are thought to be nicotinamide adenine

dinucleotide, flavin adenine dinucleotide, and porphyrins (23). There may also be a
contribution from the fluorescent oxidation adduct NFK (8).
Despite its drawbacks, SAF may have useful applications. Increased SAF observed in
dialysis patients declined after renal transplantation (24). In hemodialysis patients, SAF

was an independent predictor of overall and cardiovascular mortality. Multivariate

analysis revealed that 65% of the variance in SAF was linked to independent effects of
age; dialysis and renal failure duration; presence of diabetes; triglyceride levels; and Creactive protein (25).

PROFOUND MISHANDLING OF GLYCATED,

OXIDIZED, AND NITRATED AMINO ACIDS IN
UREMIA
With the moderate decline in renal function found in pre-dialysis chronic renal failure
TOP
ABSTRACT
MEASUREMENT OF GLYCATED,...
MEASUREMENT OF GLYCATED AND...
PROTEIN CARBONYLS AND ADVANCED...
SKIN AUTOFLUORESCENCE
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CONCLUSIONS
REFERENCES

patients, renal clearance of glycation and oxidation free adducts declines such that their
plasma concentrations increase without an increase in 24-hour urinary excretion. With
further decline in renal function to end-stage renal disease and implementation of PD
therapy, high concentrations of protein glycation, oxidation, and nitration free adducts
are maintained or further increased. In PD patients, plasma glycation free adducts are
increased by a factor of up to 18. Glycation free adduct concentrations in peritoneal
dialysate increase over a 2- to 12-hour dwell time, exceeding plasma levels markedly
and suggesting that protein glycation and oxidation adducts may form in the peritoneal
cavity and that these adducts may actively be secreted across the capillary endothelium
into the peritoneal cavity during a dialysis dwell. The high concentrations of glucose
osmolyte (74 214 mmol/L) and dicarbonyls formed during heat sterilization (up to 100
200 mol/L) sustain increased glycation in the peritoneal cavity (6,2629). Glycation
adduct formation in the peritoneal cavity declines with the use of low-GDP PD fluid (6).

In uremia, protein glycation, oxidation, and nitration adduct residues increase as a

consequence of increased concentrations of dicarbonyl glycating agents and the
oxidative stress associated with the inflammatory response to uremic toxins and
interaction with PD fluids. Changes in the turnover and plasma concentration of albumin
may also affect the content of glycation, oxidation, and nitration adduct residues in
plasma protein in PD patients. However, as currently practiced, PD does not normalize
the plasma concentrations of protein glycation, oxidation, and nitration free adducts.
Newer PD fluids with improved biocompatibility may improve the elimination of glycation
free adducts. To assess this possibility, the effect of PD fluid type on 24-hour excretion
rates of glycation, oxidation, and nitration free adducts is required.

EXCRETION RATES OF GLYCATION, OXIDATION,

AND NITRATION ADDUCTS IN PD: AN INDICATOR
TOP
ABSTRACT
OF PROTEIN DAMAGE
MEASUREMENT OF GLYCATED,...
MEASUREMENT OF GLYCATED AND...
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SKIN AUTOFLUORESCENCE
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CONCLUSIONS
REFERENCES

We measured 24-hour excretion rates of protein glycation, oxidation, and nitration free
adducts in PD patients with respect to excretion rates seen in healthy human subjects
and in pre-dialysis chronic renal failure patients. Free adduct excretion rates were
increased in PD with respect to chronic renal failure patients as follows:

Glycation free adducts:

o

FL, CML, 3DG-H, argpyrimidine, and pentosidine, by a factor of 2

MG-H1, by a factor of 6

The oxidation free adduct MetSO by a factor of 20

The nitration free adduct 3-NT by a factor of 3

The large increase in MetSO may be a result of the escape of MetSO from repair in PD
by MetSO reductase activity in the kidney. That hypothesis aside, these data suggest
that dialysis therapy may be increasing protein damage in PD patients by a factor of up
to 6. These data also provide a robust measurement by which to assess the effect of
new generations of PD fluids on protein glycation, oxidation, and nitration.

TOP
ABSTRACT
MEASUREMENT OF GLYCATED,...
MEASUREMENT OF GLYCATED AND...
PROTEIN CARBONYLS AND ADVANCED...
SKIN AUTOFLUORESCENCE
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EXCRETION RATES OF GLYCATION,...
CONCLUSIONS
REFERENCES

CONCLUSIONS
The LC-MS/MS method is the "gold standard" for measuring markers of protein
glycation, oxidation, and nitrationand also dicarbonyl GDPsin PD (12,30).
With use of low-GDP PD fluids, improved PD therapy and patient survival are now
emerging... but much more can be done. By modification and functional impairment of
extracellular matrix and mesothelial and vascular cell proteins, GDPs and AGEs impair
peritoneal membrane function and exacerbate vascular disease. Assessment of
excretion rates of glycation, oxidation, and nitration free adducts from PD patients may
provide a robust measurement for assessing the decrease in protein damage that new
generations of PD fluids are striving to achieve.

ACKNOWLEDGMENTS

The authors thank the Wellcome Trust (U.K.) and the British Heart Foundation (U.K.) for
their support for research on protein damage and vascular disease.
TOP
ABSTRACT
MEASUREMENT OF GLYCATED,...
MEASUREMENT OF GLYCATED AND...
PROTEIN CARBONYLS AND ADVANCED...
SKIN AUTOFLUORESCENCE
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CONCLUSIONS
REFERENCES

REFERENCES
1

Marrn B, Remn C, Prez-Fontn M, Quirs P, Ortz A. Benefits of preserving residual

renal function in peritoneal dialysis. Kidney Int Suppl2008; (108): S42-51.

Thornalley PJ. Dicarbonyl intermediates in the Maillard reaction. Ann N Y Acad Sci
2005;1043 : 111-17.[Medline]

Linden T, Cohen A, Deppisch R, Kjellstrand P, Wieslander A. 3 4-Dideoxyglucosone-3ene (3 4-DGE): a cytotoxic glucose degradation product in fluids for peritoneal dialysis.
Kidney Int2002; 62:697 -703.[Medline]

Morgan LW, Wieslander A, Davies M, Horiuchi T, Ohta Y, Beavis MJ, et al. Glucose
degradation products (GDP) retard remesothelialization independently of D-glucose
concentration. Kidney Int 2003; 64:1854 -66.[Medline]

Dobler D, Ahmed N, Song LJ, Eboigbodin KE, Thornalley PJ. Increased dicarbonyl
metabolism in endothelial cells in hyperglycemia induces anoikis and impairs
angiogenesis by RGD and GFOGER motif modification. Diabetes 2006; 55:1961 -9.
[Abstract/Free Full Text]

Mortier S, Faict D, Schalkwijk CG, Lameire NH, De Vriese AS. Long-term exposure to
new peritoneal dialysis solutions: effects on the peritoneal membrane. Kidney Int
2004;66 : 1257-65.[Medline]

Woywodt A, Kirsch T, Haubitz M. Circulating endothelial cells in renal disease: markers

and mediators of vascular damage. Nephrol Dial Transplant 2008; 23:7 -10.
[Free Full Text]

Ahmed N, Thornalley PJ. Advanced glycation endproducts: what is their relevance to

diabetic complications? Diabetes Obes Metab 2007; 9:233 -45.[Medline]

Thornalley PJ. Protein oxidation marker residues and oxidised amino acids in disease
the damage and the debris of protein oxidation. In: Pandalai SG, Pietzsch J, eds. Protein
Oxidation and Disease. Trivandrum, Kerala, India: Research Signpost; 2006:143 -78.

Drge W. Free radicals in the physiological control of cell function. Physiol Rev 2002;82 :
47-95.[Abstract/Free Full Text]

Thornalley PJ, Battah S, Ahmed N, Karachalias N, Agalou S, BabaeiJadidi R, et al.

Quantitative screening of advanced glycation endproducts in cellular and extracellular
proteins by tandem mass spectrometry. Biochem J 2003;375 : 581-92.[Medline]

Agalou S, Ahmed N, BabaeiJadidi R, Dawnay A, Thornalley PJ. Profound mishandling

of protein glycation degradation products in uremia and dialysis. J Am Soc Nephrol
2005;16 : 1471-85.[Abstract/Free Full Text]

Ahmed N, Argirov OK, Minhas HS, Cordeiro CA, Thornalley PJ. Assay of advanced
glycation endproducts (AGEs): surveying AGEs by chromatographic assay with
derivatisation by aminoquinolyl-N-hydroxysuccimidyl-carbamate and application to N carboxymethyl-lysine- and N -(1-carboxyethyl)lysine-modified albumin. Biochem J
2002;364 : 1-14.[Medline]

Dobler D, Ahmed N, Thornalley PJ. Peptide mapping of type IV collagen modified

minimally by methylglyoxal in vitro [Abstract]. Ann N Y Acad Sci 2005;1043 : 906.

Ikeda K, Higashi T, Sano H, Jinnouchi Y, Yishida M, Araki T, et al. N (Carboxymethyl)lysine protein adduct is a major immunological epitope in proteins
modified with advanced glycation end products of the Maillard reaction.
Biochemistry1996; 35:8075 -83.[Medline]

Koito W, Araki T, Horiuchi S, Nagai R. Conventional antibody against N (carboxymethyl)lysine (CML) shows cross-reaction to N -(carboxyethyl)lysine (CEL):
immunochemical quantification of CML with a specific antibody. J Biochem 2004;
136:831 -7.[Abstract/Free Full Text]

Rabbani N, Thornalley PJ. Assay of 3-nitrotyrosine in tissues and body fluids by liquid
chromatography with tandem mass spectrometric detection. Methods Enzymol 2008;440
: 337-59.[Medline]

Sell DR, Strauch CM, Shen W, Monnier VM. 2-Aminoadipic acid is a marker of protein
carbonyl oxidation in the aging human skin: effects of diabetes, renal failure and sepsis.
Biochem J2007; 404:269 -77.[Medline]

Requena JR, Chao CC, Levine RL, Stadtman ER. Glutamic and aminoadipic
semialdehydes are the main carbonyl products of metal-catalyzed oxidation of proteins.
Proc Natl Acad Sci U S A2001; 98:68 -74.

Kadiiska MB, Gladen BC, Baird DD, Germolec D, Graham LB, Parker CE, et al.
Biomarkers of oxidative stress study II: are oxidation products of lipids, proteins, and
DNA markers of CCl4 poisoning? Free Radic Biol Med 2005;38 : 698-710.[Medline]

WitkoSarsat V, Friedlander M, Khoa TN, CapeillereBlandin C, Nguyen AT, Canteloup

S, et al. Advanced oxidation protein products as novel mediators of inflammation and
monocyte activation in chronic renal failure. J Immunol1998; 161:2524 -32.
[Abstract/Free Full Text]

Boulanger E, Moranne O, Wautier MP, WitkoSarsat V, DescampsLatscha B,

Kandoussi A, et al. Changes in glycation and oxidation markers in patients starting
peritoneal dialysis: a pilot study. Perit Dial Int 2006;26 : 207-12.[Abstract/Free Full Text]

Na RH, Stender IM, Ma LX, Wulf HC. Autofluorescence spectrum of skin: component
bands and body site variations. Skin Res Technol 2000; 6:112 -17.[Medline]

Hartog JW, de Vries AP, Lutgers HL, Meerwaldt R, Huisman RM, van Son WJ, et al.
Accumulation of advanced glycation end products, measured as skin autofluorescence,
in renal disease. Ann N Y Acad Sci 2005; 1043:299 -307.[Medline]

Meerwaldt R, Lutgers HL, Links TP, Graaff R, Baynes JW, Gans ROB, et al. Skin
autofluorescence is a strong predictor of cardiac mortality in diabetes. Diabetes
Care2007; 30:107 -12.[Abstract/Free Full Text]

Friedlander MA, Wu YW, Elgawish A, Monnier VM. Early and advanced glycosylation
end products. Kinetics of formation and clearance in peritoneal dialysis. J Clin Invest
1996;97 : 728-35.[Medline]

Nakamura S, Miyazaki S, Sakai S, Morita T, Hirasawa Y, Niwa T. Localization of

imidazolone in the peritoneum of CAPD patients: a factor for a loss of ultrafiltration. Am
J Kidney Dis2001; 38(Suppl 1):S107 -10.[Medline]

Honda K, Nitta K, Horita S, Yumura W, Nihei H, Nagai R, et al. Accumulation of

advanced glycation end products in the peritoneal vasculature of continuous ambulatory
peritoneal dialysis patients with low ultra-filtration. Nephrol Dial Transplant1999; 14:1541
-9.[Abstract/Free Full Text]

Owen WF, Lew NL, Liu Y, Lowrie EG, Lazarus JM. The urea reduction ratio and serum
albumin concentration as predictors of mortality in patients undergoing hemodialysis. N
Engl J Med1993; 329:1001 -6.[Abstract/Free Full Text]

Thornalley PJ. Protein and nucleotide damage by methylglyoxal in physiological systems

role in ageing and disease. Drug Metabol Drug Interact 2008;23 : 125-50.[Medline]