Perit Dial Int
29(Supplement_2):
51-56
2009
© 2009 International Society for Peritoneal Dialysis
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{at}warwick.ac.uk
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ABSTRACT
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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-(2,3,4-trihydroxybutyl)-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.)
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MEASUREMENT OF GLYCATED, OXIDIZED, AND NITRATED PROTEINS AND RELATED FREE ADDUCTS BY STABLE ISOTOPIC DILUTION ANALYSIS
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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.
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 µg protein and 25 µL 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.
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MEASUREMENT OF GLYCATED AND NITRATED PROTEINS AND RELATED FREE ADDUCTS BY IMMUNOASSAY
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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).
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PROTEIN CARBONYLS AND ADVANCED PROTEIN OXIDATION PRODUCTS AS MARKERS OF PROTEIN OXIDATION
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"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-2-aminovaleric 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).
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SKIN AUTOFLUORESCENCE
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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 50-second 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
hydro-imidazolone 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 C-reactive protein
(25).
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PROFOUND MISHANDLING OF GLYCATED, OXIDIZED, AND NITRATED AMINO ACIDS IN UREMIA
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With the moderate decline in renal function found in pre-dialysis chronic
renal failure 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 µmol/L) sustain increased glycation in the peritoneal cavity
(6,26–29).
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.
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EXCRETION RATES OF GLYCATION, OXIDATION, AND NITRATION ADDUCTS IN PD: AN INDICATOR OF PROTEIN DAMAGE
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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:
- 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.
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CONCLUSIONS
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The LC-MS/MS method is the "gold standard" for measuring
markers of protein glycation, oxidation, and nitration—and also
dicarbonyl GDPs—in 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.
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ACKNOWLEDGMENTS
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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.
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ABSTRACT
MEASUREMENT OF GLYCATED,...
