In vivo method for determination of oxidative stress

A non-invasive method for the determination of oxidative stress in a patient by urinalysis is disclosed. The method comprises quantifying the level of o,o'-dityrosine in a sample of the urine of said patient and comparing with the corresponding level of said compound in a normal or control sample, whereby a substantially elevated level of said o,o'-dityrosine is indicative of oxidative stress in said patient.

FIELD OF THE INVENTION 
The present invention relates to a non-invasive method for the 
determination of oxidative stress in a patient by urinalysis. 
BACKGROUND OF THE INVENTION 
[Note: Literature references for the following background information and 
on conventional test methods and laboratory procedures well known to the 
ordinary person skilled in the art, and other such state-of-the-art 
techniques as used herein, are indicated by reference numbers in 
parenthesis and appended at the end of the specification.] 
Oxidative modification of biomolecules may contribute to the pathogenesis 
of disorders ranging from atherosclerosis to diabetes to 
ischemia-reperfusion injury (1-3). It also may play a role in the aging 
process itself (4,5). Important targets for oxidation include proteins, 
which play fundamental roles as biological catalysts, gene regulators, and 
structural components of cells (6-9). A well-characterized pathway for 
generating oxidants involves the NADPH oxidase of phagocytes. This 
membrane-associated electron transport chain produces superoxide 
(O.sub.2.sup..cndot.-) (10). 
EQU NADPH+O.sub.2 .fwdarw.NADP.sup.+ +O.sub.2.sup..cndot.- 
The superoxide then dismutates to form hydrogen peroxide (H.sub.2 O.sub.2), 
which serves as an oxidizing substrate for myeloperoxidase, a heme protein 
secreted by activated phagocytes (6-9). 
The NADPH oxidase system plays a key role in host defenses against 
microbial pathogens, and its importance is illustrated by clinical 
features of chronic granulomatous disease (CGD). In this genetic disorder, 
defects in components of the oxidase impair O.sub.2.sup..cndot.- 
production, rendering patients vulnerable to recurrent bacterial and 
fungal infections (6-9,11,12). Mice made deficient in NADPH oxidase 
simulate the CGD phenotype (13). 
Although oxidants generated by phagocytes are critical to host defenses, 
they may also damage tissue at sites of inflammation (1,14). It has been 
difficult to evaluate their pathogenic roles, however, because many of the 
current methods are nonspecific and prone to artifacts. In contrast, 
analyzing normal and diseased tissue for specific markers has proved to be 
a powerful approach to studying oxidative damage in vivo (15). Such 
markers have been identified in vitro by searching for stable products of 
protein oxidation. For example, oxidatively damaged proteins may contain 
o,o'-dityrosine cross-links, which can be generated through a variety of 
mechanisms. These may involve peroxynitrite, nitrogen dioxide radical, 
hydroxyl radical, or the myeloperoxidase-peroxide system of activated 
phagocytes, which cross-links tyrosine residues (16-19). Tissue levels of 
o,o'-dityrosine are elevated in atherosclerosis, exercise, Alzheimer's 
disease, and aging (20-23). Oxidative stress has been linked to all of 
these conditions. 
Dityrosine also may mark oxidatively damaged proteins for proteolytic 
destruction because red blood cells exposed to H.sub.2 O.sub.2 release 
this abnormal amino acid, suggesting that its formation in proteins may 
lead to protein breakdown (24). 
The present inventor has proposed that oxidized amino acids might be 
excreted from cells rather than being re-used for protein synthesis. After 
entering the blood, they would be filtered by the kidney into urine. The 
demonstrations in the priority applications, Ser. No. 60,074,167 and 
09/170,513, that levels of urinary o,o'-dityrosine are elevated in 
exercised and aging animals and that antioxidant therapy decreases these 
levels, is consistent with this proposal (21,22). 
BRIEF DESCRIPTION OF THE INVENTION 
In accordance with the present invention, a non-invasive method for the 
determination of oxidative stress in a patient by urinalysis is provided. 
The method comprises quantifying the level of o,o'-dityrosine in a sample 
of the patient's urine and comparing with the corresponding level in a 
normal or control sample. A substantially elevated level of that amino 
acid is indicative of oxidative stress in the patient. 
The finding of a substantially elevated level of o,o'-dityrosine in the 
urine of a patient subject to oxidative stress was unexpected since amino 
acids are normally broken down or resorbed during the usual metabolic 
processes in the body for several reasons: 
First, amino acids are generally known to be almost completely resorbed 
from glomular filtrate in the proximal tubule of the kidney. See, for 
example, TEXTBOOK OF MEDICAL PHYSIOLOGY, by Arthur C. Guyton, M.D., Eighth 
Edition, 1991, W. B. Saunders Company, pp. 303-304. 
Secondly, the oxidized amino acid, o,o'-dityrosine, is shown herein to be 
excreted from the blood into urine almost quantitatively. This is a 
critical finding in accordance with the present invention for providing a 
non-invasive method for the determination of oxidative stress in a 
patient. If, instead, only a minor fraction of the amino acid were 
excreted into urine, changes in the excretion rate (or metabolism rate; 
see below) rather than oxidative stress might account for changes in urine 
level. 
The observation that dityrosine is excreted nearly quantitatively was 
unexpected for several reasons. First, as noted above, it is surprising 
that dityrosine itself is excreted in vivo in urine at all. Second, this 
indicates that dityrosine injected intravenously is not metabolized to 
other compounds. Most amino acids transfer their amino groups (which are 
an important cellular nutrient) to acceptor molecules when they are 
degraded. See, for example, TEXTBOOK OF MEDICAL PHYSIOLOGY, id, at page 
768. 
A striking example of the typical metabolic fate of amino acids is observed 
with the oxidized amino acid 3-nitrotyrosine. Minimal amounts of this 
amino acid (a marker of reactive nitrogen species) are excreted into urine 
when 3-nitrotyrosine is given to animals. See, for example, Tabrizi-Fard, 
et al., Drug Metab. Dispos. 27(4), 429-431 (1999). Instead, metabolites of 
the amino acid such as 3-nitro-4-hydroxyphenylacetic acid, 
3-nitro-4-hydroxyphenyllic acid, and possibly other species appear in 
urine. Tabrizi-Fard, id, and Ohshimi et al., Food Chem. Toxicol. 28(9), 
647-652 (1990). 
Collectively, these findings indicate that it is not obvious that 
dityrosine would be quantitatively excreted in urine, and they support the 
inventor's contention that quantifying the level of o,o'-dityrosine in 
urine is a useful, non-invasive and unobvious method to assess oxidative 
stress in a patient. 
DETAILED DESCRIPTION OP THE INVENTION 
While the specification concludes with claims particularly pointing out and 
distinctly claiming the subject matter regarded as forming the present 
invention, it is believed that the invention will be better understood 
from the following preferred embodiments taken in conjunction with the 
accompanying drawings.

In order to illustrate the invention in greater detail, the following 
specific laboratory examples were carried out. Although specific examples 
are thus illustrated herein, it will be appreciated that the invention is 
not limited to these specific examples or the details therein. 
In these Examples, it was first determined whether phagocyte activation 
promotes oxidative tissue damage by determining levels of o,o'-dityrosine 
in inflammatory cells isolated from the peritoneum of wild-type and CGD 
mice. Isotope dilution gas chromatography/mass spectrometry (GC/MS), a 
sensitive and specific method, was used in these Examples. Levels of 
o,o'-dityrosine rose transiently in the proteins of the leukocytes of the 
wild-type animals, as did the amount of o,o'-dityrosine excreted in urine. 
These increases failed to occur in the CGD mice, indicating that protein 
oxidation under these in vivo conditions requires the participation of the 
NADPH oxidase of activated phagocytes. These results indicate that 
oxidized tissue proteins undergo proteolysis and that the resulting free 
o,o'-dityrosine is excreted in urine (4,24-26). They strongly support the 
present contention that oxidized amino acids in urine represent a 
noninvasive marker of oxidative stress in vivo. Moreover, a striking 
increase in urinary o,o'-dityrosine was detected in patients with systemic 
inflammation. Clinical tests based on such a marker would be invaluable 
for the assessment of oxidative stress in human disease. 
EXAMPLES 
Materials. 
Unless otherwise indicated, reagents were obtained from Sigma Chemical 
Company (St. Louis, Mo.) or Aldrich Chemical (Milwaukee, Wis.). Organic 
solvents were HPLC grade. 
Animal Studies. 
The animal studies committee of Washington University School of Medicine in 
St. Louis approved all protocols. The C57BL/6J mice used for these 
experiments were obtained from Jackson Laboratories (Bar Harbor, Mass.) 
and bred and maintained under pathogen-free conditions. Impaired 
O.sub.2.sup..cndot.- by phorbol ester-stimulated neutrophils isolated from 
CGD mice deficient in gp91 phox (13) was confirmed by monitoring the 
superoxide dimutase inhibitable reduction of ferricytochrome c (10). Mice 
were fed rodent diet 5001 (Harlan-Teklad, Madison, Wis.). Between ages 9 
and 11 weeks, the mice were injected with one ml of 4% thioglycollate to 
recruit leukocytes into the peritoneum. The leukocytes were activated 20 
hours (h) later by intraperitoneal injection of zymosan (250 mg/kg). 
Control animals were injected with one ml of buffer A (138 mM NaCl, 3 mM 
KCl, 0.1 M sodium phosphate, pH 7.4). Prior to and every 2 h during urine 
collection, the animals were injected intraperitoneally with 2 ml of 
buffer A. 
Urine Collection. 
Animals were placed in stainless steel metabolic cages designed for urine 
collection (21). All subsequent procedures were performed under subdued 
light. Urine was collected in amber-colored vials containing 40 .mu.l of 
500 .mu.M diethylenetriaminepentaacetic acid (DTPA, a metal chelator) and 
4 .mu.l of 5% phenol (an antioxidant and bactericidal agent). Collected 
urine was adjusted to a final concentration of 100 .mu.M DTPA and 0.1% 
phenol and stored at -80.degree. C. until analysis. Creatinine levels of 
urine were measured using Sigma Diagnostic Kit 555-A. 
Isolation of Amino acid from Urine. 
Urine was centrifuged at 10,000.times.g for 5 min. An aliquot of the urine 
supernatant (50 .mu.L for mouse and 100 .mu.l for human samples) was 
supplemented with 0.1% trifluoroacetic acid (TFA; v/v; final volume 200 
.mu.l) and .sup.13 C internal standards. Amino acids were isolated by 
solid-phase extraction on a C-18 column (3 ml; Supelclean SPE; Supelco, 
Bellefonte, Pa.) conditioned with 4 ml of methanol and 6 ml of 0.1% TFA. 
The column was loaded with urine in 0.1% TFA and washed with 2 ml of 0.1% 
TFA using a vacuum manifold system. Amino acids were eluted with 2 ml of 
50% methanol and concentrated to dryness under vacuum for derivatization. 
Recovery of authentic o,o'-dityrosine subjected to this procedure was&gt;90%. 
Leukocyte Harvesting. 
Animals were killed by decapitation after metophane-induced anesthesia. 
After the abdominal wall was washed with 95% ethanol, leukocytes were 
harvested from the peritoneum using a siliconized glass pipette. The 
peritoneum was lavaged 3 times with 4 ml of ice-cold Hanks' Balanced Salt 
Solution (HBSS) containing 100 .mu.M DTPA. Cells were suspended in 
ice-cold HBSS, collected by low speed centrifugation, and stored at 
-80.degree. C. until analysis. 
Isolation of Amino Acids from Leukocytes. 
All procedures were carried out at 4.degree. C. Peritoneal cells were 
dialyzed against antioxidant buffer (100 .mu.M DTPA, 100 .mu.M butylated 
hydroxytoluene, 10 mM 3-aminotriazole, 10 mM sodium phosphate, pH 7) and 
0.1 mM DTPA (pH 7) and delipidated 3 times with 
water/methanol/water-washed ether (1:3:7; v/v/v). Residual solvent was 
removed under vacuum, .sup.13 C labeled internal standards were added, and 
protein was hydrolyzed in one ml of 6 N HCl containing 1% phenol at 
110.degree. C. for 24 h under argon. Amino acids were isolated from the 
acid hydrolysate by solid-phase extraction as described above. 
Mass Spectrometric Analysis. 
Amino acids were converted to their n-propyl esters, heptafluorobutyryl 
derivatives and quantified by isotope dilution gas chromatography/mass 
spectrometry (GC/MS) with selected ion monitoring (20,21). Mass 
spectrometric analysis was performed in the negative ion electron capture 
mode with methane as the reagent gas, using a Finnigan SSQ with extended 
mass range equipped with a 12 m DB-1 capillary column (0.2 mm ID, 0.33 
.mu.m film thickness; J&W Scientific, Folson, Calif.). The ions monitored 
were: L-tyrosine, m/z 417 and 595; L-[.sup.13 C.sub.6 ]tyrosine, m/z 423 
and 601; o,o'-dityrosine, m/z 1030 and 1208; o,o'-[.sup.13 C.sub.12 
]dityrosine, m/z 1042 and 1220. Under these conditions, authentic 
compounds and isotopically labeled standards were baseline separated and 
exhibited retention times identical to those of analytes. 
Metabolic fate of radiolabeled dityrosine. 
o,o'-[.sup.14 C]Dityrosine (2.times.10.sup.5 cpm) was injected into the 
animals intravenously. Plasma was centrifuged (400.times.g for 20 min at 
4.degree. C.) from blood collected by retro-orbital bleeding. Tissue 
samples were collected from animals killed by cervical dislocation, 
weighed and then homogenized in deionized water. Radioactivity in tissue 
homogenates, plasma, and urine was determined by liquid scintillation 
spectroscopy (Beckman LS 3801) with correction for quenching. [.sup.14 
C]Dityrosine was prepared from L-[.sup.14 C]tyrosine (American 
Radiolabeled Chemicals Co.; St. Louis, Mo.) and o,o'-[.sup.13 C.sub.12 
]dityrosine was prepared from L-[.sup.13 C.sub.6 ]tyrosine (Cambridge 
Isotope Inc.; Andover, Mass.) using horseradish peroxidase (Boehringer 
Mannheim Biochemicals; Indianapolis, Ind.) and H.sub.2 O.sub.2 (Fisher 
Scientific Co.; Pittsburgh, Pa.) and isolated by reverse-phase HPLC; (27). 
o,o'-[.sup.14 C]Dityrosine and radiolabeled material in urine were 
analyzed by HPLC (27) using a reverse-phase column (ODS ultrasphere, 250 
mm.times.4.6 mm, particle size 5 .mu.m.; Beckman,) and a flow rate of 1 
ml/min. The column was equilibrated with 100% solvent A (1% acetic acid in 
water). Compounds were eluted with a discontinuous gradient of solvent B 
(1% acetic acid in methanol). The gradient was 0%-10% solvent B over 10 
min, 10% solvent B for 4 min, 10%-100% solvent B over 5 min and 100% 
solvent B for 10 min. Prior to HPLC analysis, urinary amino acids were 
isolated by solid-phase extraction as outlined above. Recovery of 
radiolabeled material from urine subjected to this procedure was&gt;90%. 
Radioactivity in each HPLC fraction was quantified by liquid scintillation 
spectroscopy. 
Results 
To determine whether white blood cells oxidatively damage proteins in vivo, 
isotope dilution GC/MS was used to quantify levels of o,o'-dityrosine in 
mouse peritoneal phagocytes. To recruit neutrophils into the peritoneum, 
the animals were injected with thioglycollate, a chemical irritant. Twenty 
hours after this first injection, the animals were given an 
intraperitoneal injection of zymosan, which stimulates phagocytes to 
produce H.sub.2 O.sub.2 and secrete myeloperoxidase into the phagolysosome 
(6). Thioglycollate-injected mice typically yielded 20-40.times.10.sup.6 
peritoneal cells. Neutrophils, mononuclear cells, and eosinophils 
represented 67.+-.4%, 30.+-.4%, and&lt;1% of the harvested phagocytes (n=6 
animals). 
Acute inflammation increases o,o'-dityrosine levels in urine. 
To determine whether animals that develop acute inflammation increase their 
excretion of o,o'-dityrosine, levels of the oxidized amino acid in urine 
were measured by negative ion electron capture GC/MS with selected ion 
monitoring. To correct for individual differences in glomerular 
filtration, levels of o,o'-dityrosine were normalized to levels of urinary 
creatinine. The concentration of o,o'-dityrosine was 3-fold greater in the 
urine of mice injected with thioglycollate and zymosan than in control 
animals or animals injected with thioglycollate alone (FIG. 1). The 
increase was maximal 12 h after zymosan injection, and it declined to the 
baseline level over the next 12 h (FIG. 1A). Urinary o,o'-dityrosine 
failed to increase in CGD mice injected intraperitoneally with 
thioglycollate and zymosan (FIG. 1B). These results indicate that 
degradation of oxidatively damaged proteins in phagocytes releases free 
o,o'-dityrosine that enters the blood and is excreted in urine. 
To investigate whether inflammation might alter the rate at which the 
kidneys excrete either creatinine or the precursor amino acid of 
o,o'-dityrosine, levels of creatinine and tyrosine in urine were 
quantified. There were no differences in creatinine among the animals 
subjected to the different regimens. The ratio of tyrosine to creatinine 
in the urine was similar in all groups, suggesting that differential amino 
acid excretion was not responsible for the differences in o,o'-dityrosine 
levels that were observed in the urine samples. Moreover, the kinetics and 
magnitude of o,o'-dityrosine output into the urine of wild-type mice 
injected with thioglycollate and zymosan were similar when o,o'-dityrosine 
levels were normalized to urine levels of tyrosine. These results indicate 
that alterations in renal excretion of creatinine or amino acids do not 
account for the elevated o,o'-dityrosine levels observed in the urine of 
wild-type mice subjected to acute inflammation. 
Acute inflammation elevates o,o'-dityrosine levels in phagocyte proteins. 
Resident thioglycollate-elicited neutrophils contained .about.50 .mu.mol 
o,o'-dityrosine per mol tyrosine. When neutrophils were exposed to 
zymosan, however, levels of o,o'-dityrosine in cellular proteins increased 
4-fold (FIG. 2A). This increase was transient, peaking at 3 h. Twelve 
hours after the zymosan injection, the level of protein-bound dityrosine 
in peritoneal neutrophils was similar to those observed in non-activated 
neutrophils. These results suggest that activating phagocytes with zymosan 
transiently increases protein oxidation. The subsequent decline in 
protein-bound dityrosine may reflect intracellular degradation of oxidized 
proteins in neutrophils, migration of activated leukocytes out of the 
peritoneum, or phagocytosis of apoptotic or activated neutrophils by 
macrophages. 
To determine whether reactive oxygen species generated by the NADPH oxidase 
damage cellular proteins, levels of o,o'-dityrosine were compared after 3 
hours of zymosan-stimulation in phagocytes harvested from wild-type and 
CGD mice (FIG. 2B). The increase in protein-bound dityrosine was 
completely abrogated in mice deficient in NADPH oxidase (p&lt;0.0005). 
Moreover, o,o'-dityrosine was undetectable in the proteins of these cells, 
suggesting that oxidants generated by the NADPH oxidase represent the 
predominant pathway for generation of the tyrosine cross-link in 
phagocytes. Collectively, these observations indicate that 
zymosan-stimulated phagocytes oxidatively damage proteins in vivo by a 
pathway that requires O.sub.2.sup..cndot.- and/or H.sub.2 O.sub.2. 
Radiolabeled o,o'-dityrosine is rapidly cleared from the blood and is 
recovered in near-guantitative yield in urine. 
To confirm that free o,o'-dityrosine is excreted in urine and to study the 
fate of the oxidized amino acid in the body, o,o'-[.sup.14 C]dityrosine 
was injected intravenously into mice and the amount of radioactivity that 
subsequently appeared in blood, tissue, and urine samples was measured 
(FIG. 3). Dityrosine was cleared from the blood with biphasic kinetics 
(FIG. 3A). It initially disappeared rapidly, with more than 50% of the 
radiolabeled material lost 5 min after injection. Its subsequent 
disappearance was slower, with an apparent half-life in the blood of 
.about.30 min. Thirty percent of the radioactivity was recovered in the 
urine 1 h after intravenous injection of o,o'-[.sup.14 C]dityrosine, 65% 
was recovered after 3 h, and 81% was recovered after 4 h (FIG. 3B). In 
striking contrast, radiolabeled material was essentially undetectable in 
blood, liver, spleen, and heart. Small amounts (&lt;5%) were present in 
kidney, perhaps due to urine in the renal collecting system. These results 
indicate that free o,o'-dityrosine is rapidly cleared from blood and 
quantitatively recovered in urine, strongly suggesting that the oxidized 
amino acid is filtered from the blood by the kidneys and excreted in 
urine. They also suggest that cells do not take up o,o'-dityrosine from 
blood and incorporate it into proteins to a significant degree. 
To determine whether any of the o,o'-dityrosine that was injected 
intravenously was metabolized prior to excretion, the radiolabeled 
material that appeared in urine was subjected to reverse-phase HPLC 
analysis (FIG. 4). Previous studies have shown that this chromatography 
system readily separates a wide range of tyrosine oxidation products (27). 
More than 80% of the radioactivity in the present samples co-migrated with 
authentic o,o'-[.sup.14 C]dityrosine. These results indicate that most of 
the radiolabeled material that was injected into the animals and recovered 
from urine was free o,o'-[.sup.14 C]dityrosine under the analytical 
conditions used herein. The remaining radiolabeled material likely 
represents metabolites derived from o,o'-[.sup.14 C]dityrosine. 
The urine of septic humans is enriched in o,o'-ditvrosine. 
To confirm the utility of o,o'-dityrosine as a clinical marker of oxidative 
stress, isotope dilution GC/MS was used to quantify the oxidized amino 
acid in human urine (FIG. 5). Selected ion monitoring demonstrated that 
the negative ions derived from derivatized o,o'-dityrosine co-eluted with 
ions derived from authentic .sup.13 C-labeled internal standards in these 
samples (FIG. 5A). Urine from acutely ill patients with sepsis (fever, 
elevated white blood cell count and a known source of infection) had a 
three-fold increase (FIG. 5B, p&lt;0.005) in the concentration of 
o,o'-dityrosine compared with urine from young, healthy subjects (20 to 40 
years old). These results indicate that healthy humans excrete 
o,o'-dityrosine in urine and that levels of the oxidized amino acid are 
markedly elevated in septic patients. 
In the above examples, isotope dilution GC/MS was used to simultaneously 
quantify levels of o,o'-dityrosine in both inflammatory cells and urine of 
wild-type and CGD mice. The use of CGD mice allowed assessment of the role 
of phagocyte NADPH oxidase in promoting protein oxidation in vivo. 
o,o'-Dityrosine levels were monitored for four reasons. 
First, previous in vitro studies have shown that activated phagocytes 
produce oxidants that generate o,o'-dityrosine by pathways that involve 
tyrosyl radical generated by myeloperoxidase, reactive nitrogen species, 
or hydroxyl radical (16-19,28). 
Second, elevated levels of protein-bound dityrosine--a specific marker of 
oxidation--have been detected in various pathological states, suggesting 
that tyrosyl radical may be one agent that promotes oxidative stress in 
vivo. 
Third, in vitro studies indicate that o,o'-dityrosine serves as a marker 
for the proteolysis of oxidized proteins in red blood cells exposed to 
H.sub.2 O.sub.2 (24). 
Finally, it was believed that this cross-linked amino acid was likely to be 
excreted from the body in urine rather than used to synthesize new 
proteins. 
The above results indicate that stimulating phagocytes with zymosan in vivo 
markedly increases their content of protein-bound dityrosine. This 
increase failed to occur in CGD mice, implicating phagocyte activation and 
the NADPH oxidase in protein oxidation (1,14,16-18,28,29). The level of 
phagocyte o,o'-dityrosine peaked 3 h after the cells were activated; it 
then rapidly returned to that observed in quiescent phagocytes. 
Importantly, the concentration of o,o'-dityrosine in urine also increased 
in this mouse model of acute inflammation. Again, this increase was 
inhibited in CGD mice. Urinary o,o'-dityrosine levels peaked 12 h after 
stimulation of the cells with zymosan, following the transient increase in 
phagocyte o,o'-dityrosine levels. Collectively, these observations 
indicate that activated phagocytes oxidatively modify proteins during 
inflammation by a pathway dependent upon NADPH oxidase, implicating 
O.sub.2.sup..cndot.- and/or H.sub.2 O.sub.2 in the reaction. The kinetics 
of its appearance in cells and then urine strongly confirm that 
oxidatively modified proteins undergo proteolysis and that the liberated 
o,o'-dityrosine is excreted in urine. 
o,o'-Dityrosine was detected in the urine of healthy humans, but the 
concentration was three times as high in the urine of patients suffering 
from systemic bacterial infections. Bacteria are known to activate 
phagocytes, whose NADPH oxidase plays a key role in host defenses against 
invading pathogens (1,6,14). This indicates that oxidants generated by 
phagocytes are one physiologically relevant pathway for protein oxidation 
in humans. Therefore, assaying o,o'-dityrosine is a useful approach to 
monitoring oxidative stress in vivo. 
It is noteworthy that o,o'-dityrosine is present in the tissues and urine 
of healthy young humans and control animals. This indicates that pathways 
not involving phagocyte NADPH oxidase also can generate this oxidized 
amino acid. Heme proteins and hydroxyl radical-like species generated by 
mitochondria represent other mechanisms for o,o'-dityrosine formation in 
vivo (21,22). The use of mice with genetically engineered alterations in 
these pathways provides a powerful test of this mechanism. 
An important point regarding the use of o,o'-dityrosine to monitor 
oxidative stress in vivo is the fate of the cross-linked amino acid in the 
body. For example, o,o'-dityrosine might be re-incorporated into proteins 
or metabolized to other compounds, invalidating the relationship between 
its concentration in urine and protein oxidation. This issue was addressed 
by injecting purified radiolabeled o,o'-dityrosine intravenously into mice 
and monitoring the disappearance of the oxidized amino acid from blood. 
More than half of the radiolabel disappeared from the blood in 5 min, 
indicating that o,o'-dityrosine is taken up by tissues or excreted into 
the urine by the kidney. Between 3 h and 4 h later, the radioactive 
material was recovered almost quantitatively in the urine. Analysis by 
reverse-phase HPLC with a gradient that separates a wide variety of 
tyrosine oxidation products (27) indicated that most of the radioactivity 
comigrated with authentic o,o'-dityrosine. These results indicate that 
o,o'-dityrosine released from proteins is excreted in urine in 
near-quantitative yield and that the oxidized amino acid in blood is 
relatively resistant to metabolism into other compounds. 
These in vivo results suggest the following model. Activated phagocytes use 
oxidants produced by the NADPH oxidase to generate tyrosyl radical or 
other reactive species that form o,o'-dityrosine cross-links in proteins. 
The oxidized proteins then are selectively targeted for proteolytic 
degradation (4,24-26). Alternatively, macrophages may phagocytose and 
digest neutrophils that have been activated and are undergoing apoptosis 
(30,31). The resulting free oxidized amino acids are released from cells, 
enter the blood, are filtered out of the blood by the kidneys, and 
excreted in near-quantitative yield in the urine. This model is consistent 
with the above results whereby quantifying o,o'-dityrosine and other 
oxidized amino acids in urine may provide a noninvasive method for 
measuring protein oxidative stress in vivo. Such an assay is invaluable 
for assessing the role of oxidative stress in aging and the pathogenesis 
of human disease. 
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