Oxidized amino acids in the urine of aging rats:
potential markers for assessing oxidative stress in vivo
POLLY A. HANSEN,
JOHN O. HOLLOSZY,
AND JAY W. HEINECKE
Internal Medicine and
Molecular Biology and Pharmacology,
Washington University School of Medicine, St. Louis, Missouri 63110
Leeuwenburgh, Christiaan, PollyA. Hansen, John O.
Holloszy, and Jay W. Heinecke.
Oxidized amino acids in
the urine of aging rats: potential markers for assessing
oxidative stress in vivo.
Am. J. Physiol.
Integrative Comp. Physiol.
45): R128–R135, 1999.—Oxida-
tive damage of proteins has been implicated in disease and
aging. In vitro studies demonstrate that two unnatural amino
-tyrosine, are stable markers of
protein oxidation. We have investigated the possibility that
assaying these compounds in urine could provide a noninva-
sive way to determine levels of protein oxidation in vivo.
Isotope dilution gas chromatography-mass spectrometry was
used to quantify levels of
skeletal muscle and urine of aging rats subjected to two
) dietary antioxidant supplementation and
exercise training. In both sedentary rats and exercise-trained
rats, antioxidant therapy reduced levels of protein-bound
-dityrosine in skeletal muscle. In contrast, antioxidant
therapy or exercise training minimally affected
levels in this tissue. Levels of the oxidized amino acids in
urine samples mirrored those of skeletal muscle proteins.
Quantiﬁcation of the levels of oxidized amino acids in urine
may thus serve as a noninvasive measure of oxidative stress
in vivo because they change in parallel with levels of protein-
bound oxidized amino acids in skeletal muscle.
-dityrosine; ortho-tyrosine; antioxidants; exercise; protein
OXIDATIVE DAMAGE OF
proteins, lipids, and nucleic acids
has been implicated in diseases ranging from atheroscle-
rosis to ischemia-reperfusion injury to cancer (1–3, 16,
32). Many lines of evidence also suggest that such
damage plays a causal role in aging (1–3, 23, 28, 33,
34). One important target may be proteins (3, 33),
which play fundamental roles as biological catalysts,
gene regulators, and structural components of cells.
One widely studied model of protein oxidation involves
metal-catalyzed reactions that generate hydroxyl radi-
cal and other reactive species. These oxidants generate
reactive carbonyls from certain amino acid residues (3,
23, 33). The discovery of elevated levels of protein
carbonyls in many pathological states (3) and in tissues
of old animals (3, 28, 33, 34) has implicated protein
oxidation in the pathogenesis of disease and aging.
A major difficulty in evaluating the roles of oxidants
in human disease has been the lack of precise measures
of oxidative stress in vivo (7). Many of the currently
available methods are nonspeciﬁc and prone to arti-
facts. A powerful approach to studying oxidative dam-
age in vivo is the analysis of normal and diseased tissue
for speciﬁc markers (9, 21, 26, 30, 31). Such markers