U.S. patent application number 12/538854 was filed with the patent office on 2010-04-29 for methods of quantifying n2-(1-carboxyethyl)-2'-deoxy-guanosine (cedg) and synthesis of oligonucleotides containing cedg.
Invention is credited to Samuel Rahbar, Timothy W. Synold, John Termini.
Application Number | 20100102218 12/538854 |
Document ID | / |
Family ID | 42116568 |
Filed Date | 2010-04-29 |
United States Patent
Application |
20100102218 |
Kind Code |
A1 |
Rahbar; Samuel ; et
al. |
April 29, 2010 |
METHODS OF QUANTIFYING N2-(1-CARBOXYETHYL)-2'-DEOXY-GUANOSINE
(CEdG) AND SYNTHESIS OF OLIGONUCLEOTIDES CONTAINING CEdG
Abstract
Methods of quantifying a N.sup.2-carboxyethyl-2'-deoxyguanosine
(CEdG) levels in biological samples and comparing those levels to
known normal levels can diagnose a number of disorders, including
diabetes and cancer. Methods can also determine whether therapies
for disorders are effective by measuring CEdG levels before and
after treatment. Measurement of CEdG levels occurs using liquid
chromatography electrospray ionization tandem mass
spectrometry.
Inventors: |
Rahbar; Samuel; (Beverly
Hills, CA) ; Synold; Timothy W.; (Monrovia, CA)
; Termini; John; (Altadena, CA) |
Correspondence
Address: |
PERKINS COIE LLP
POST OFFICE BOX 1208
SEATTLE
WA
98111-1208
US
|
Family ID: |
42116568 |
Appl. No.: |
12/538854 |
Filed: |
August 10, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61087393 |
Aug 8, 2008 |
|
|
|
Current U.S.
Class: |
250/282 ;
73/61.52 |
Current CPC
Class: |
G01N 30/7266 20130101;
G01N 30/7233 20130101; G01N 2030/045 20130101; G01N 2030/8827
20130101; G01N 2030/8868 20130101; A61N 5/10 20130101; G01N
2800/042 20130101; A61K 31/195 20130101; A61K 45/06 20130101; A61P
35/00 20180101 |
Class at
Publication: |
250/282 ;
73/61.52 |
International
Class: |
B01D 59/44 20060101
B01D059/44; G01N 30/00 20060101 G01N030/00 |
Goverment Interests
GOVERNMENT INTEREST
[0002] The present invention was made with government support under
City of Hope's Cancer Center Support Grant (NIH Grant No. P30
CA33572) and the California Breast Cancer Research Program for a
pre-doctoral fellowship to D. Tamae (14GB-0162). The government has
certain rights in the present invention.
Claims
1. A method of quantifying an advanced glycation end product (AGE)
in a sample, comprising: (a) obtaining a biological sample from a
subject; and (b) performing liquid chromatography electrospray
ionizing tandem mass spectrometry assay on the sample using a
stable isotope dilution and an internal standard; wherein the assay
determines the quantity of AGE in the sample.
2. The method of claim 1, wherein the AGE is
N.sup.2-carboxyethyl-2'-deoxyguanosine (CEdG) and the internal
standard is .sup.15N.sub.5-CEdG.
3. The method of claim 2, wherein elevated or depressed CEdG levels
in a subject as compared to normal physiological CEdG levels
indicates the presence of a metabolic disorder in the subject.
4. The method of claim 2, wherein elevated CEdG levels in a subject
as compared to normal physiological CEdG levels indicates
diabetes.
5. The method of claim 2, wherein depressed CEdG levels in a
subject as compared to normal physiological CEdG levels indicates
cancer.
6. The method of claim 5, wherein the cancer is breast cancer.
7. The method of claim 1, wherein the biological sample is selected
from the group consisting of nucleic acid, urine, and tissue.
8. A method of detecting diabetes in a subject comprising: (a)
obtaining a biological sample from the subject; and (b) quantifying
N.sup.2-carboxyethyl-2'-deoxyguanosine (CEdG) level in the sample
using liquid chromatography electrospray ionization tandem mass
spectrometry and an internal CEdG standard; wherein, an elevated
level of CEdG indicates that the subject has diabetes.
9. The method of claim 8, wherein the method further comprises,
upon determining that a subject has diabetes, (c) administering a
treatment for diabetes; and (d) after administration, repeating
steps (a) and (b) to determine whether the treatment is
therapeutically effective, wherein if the level of CEdG has been
reduced or returned to normal, the treatment is therapeutically
effective.
10. The method of claim 8, wherein the biological sample is urine
or nucleic acid.
11. The method of claim 10, wherein the nucleic acid is
double-stranded DNA.
12. The method of claim 8, wherein the internal standard is
.sup.15N.sub.5-CEdG.
13. The method of claim 8, further comprising preventing
artifactual CEdG formation by adding aminoguanidine and/or
D-penicillamine to the sample between steps (a) and (b).
14. A method of detecting a glycolytic cancer in a subject
comprising: (a) obtaining a biological sample from the subject; and
(b) quantifying a N.sup.2-carboxyethyl-2'-deoxyguanosine (CEdG)
level in the sample using liquid chromatography electrospray
ionization tandem mass spectrometry and an internal standard of
.sup.15N.sub.5-CEdG; wherein, a depressed level of CEdG indicates
that the subject has a glycolytic cancer.
15. The method of claim 14, wherein the method further comprises,
upon determining that a subject has a glycolytic cancer, (c)
administering a treatment for the cancer; and (d) after
administration, repeating steps (a) and (b) to determine whether
the treatment is therapeutically effective, wherein if the level of
CEdG has been increased or returned to normal, the treatment is
therapeutically effective.
16. The method of claim 15, wherein the treatment for cancer is
chemotherapy or radiotherapy.
17. The method of claim 14, wherein glycolytic cancer is breast
cancer and the biological sample is breast tissue.
18. The method of claim 14, wherein the biological sample is
nucleic acid.
19. The method of claim 18, wherein the nucleic acid is
double-stranded DNA.
Description
PRIORITY CLAIM
[0001] This application claims the benefit of U.S. Provisional
Application Ser. No. 61/087,393, filed Aug. 8, 2008, which is
incorporated herein by reference.
BACKGROUND
[0003] Methylglyoxal (MG) is a highly reactive electrophile and is
present at micromolar levels in many foods and most living
organisms. MG is a major environmental breakdown product of
carbohydrates. MG is also generated biochemically during glycolysis
via elimination of phosphate from the common enediol intermediate
resulting from deprotonation of dihydroxyacetone phosphate and
glyceraldehyde 3-phosphate. Additional endogenous sources of MG
include the catabolism of threonine and the P450 mediated oxidation
of ketone bodies and the oxidative breakdown of DNA and RNA under
acidic conditions. MG is a probable mutagen in vivo.
[0004] Methylglyoxal induces G>T and G>C transversions, as
well as a large number (50%) of multibase deletions. Since 89% of
the base substitution mutations are observed at guanosine, and
N.sup.2-(1-Carboxyethyl)-2'-Deoxy-Guanosine ("CEdG") is the
predominant adduct formed from reaction of MG with DNA, this
pattern of transversions arises from CEdG (as primer extension
assays using oligonucleotide templates containing CEdG have
evidenced). The presence of CEdG in DNA has also been shown to
induce single-strand breaks, suggesting an alternative mechanism by
which this adduct may contribute to genetic instability.
[0005] Glycation results when a sugar, such as fructose or glucose,
non-enzymatically links to a protein or lipid. Glycation typically
impairs the function of the molecules to which it binds.
Methylglyoxal reacts readily with nucleophilic moieties on
proteins, lipids and DNA to produce covalent adducts known as
advanced glycation end-products (AGEs). Protein AGEs are well
characterized and these highly modified proteins have been proposed
to play a role in the various pathologies associated with diabetes,
cancer, aging, and Alzheimers disease. The first clear correlation
between abnormal levels of a protein-AGE and a human disease
(diabetes) was described in 1969 for the hemoglobin HbA.sub.1c,
adduct by Rahbar et al. Since then, hemoglobin HbA.sub.1c has
become a commonly used biomarker for the diagnosis and treatment
monitoring of diabetes..sup.11-13 Accordingly, there is continued
interest in the development of novel, more sensitive assays for the
quantitative measurement of biomolecule-derived AGEs to complement
and extend the clinical biomarker repertoire, as well as to assist
in elucidating their role in pathology.
[0006] Approximately a dozen protein-AGEs have been characterized
and LC-MS/MS methods have been described for their quantitative
measurement. Choosing an appropriate protein-AGE biomarker for
evaluating the glycation status of a particular target tissue or
organ is complicated by unequal protein-AGE distributions across
different tissues, varying adduct stabilities, and the limited
availability of stable isotope standards for quantification.
Glycation adducts of DNA have potential as biomarkers since all
nucleated cells contain the same DNA content and should reflect the
relative level of MG in the target tissue.
[0007] In spite of longstanding interest in the role of biopolymer
glycation in human disease, no generally applicable method for the
quantitative determination of CEdG has been described. A .sup.32P
post-labeling assay has been used to estimate endogenous levels of
CEdG in human buccal epithelial cells of 2-3/10.sup.7
nucleotides..sup.28 However, although the post-labeling method
offers potential advantages in sensitivity, a major drawback is
that direct analyte verification is not possible. Moreover,
post-labeling is prone to artifacts and false positives, and may
lead to inaccurate estimation of adduct levels due to several
factors including RNA contamination.
[0008] An immunoaffinity-based method for the detection of CEdG
using a polyclonal antibody coupled to a diode array HPLC platform
has more recently been described by Schneider et al in 2006. This
approach was used to provide the first demonstration of CEdG in
human urine and cultured smooth muscle cells. In some cases, peak
identity was confirmed by LC-MS/MS, but quantitation was not
practical due to the imprecise nature of immunoaffinity
chromatography. A monoclonal-based immunohistochemical detection
method has also been reported and was used to demonstrate elevated
levels of CEdG in aorta and kidney of diabetic patients relative to
normal controls..sup.31 However, antibody-based assays are
primarily of value in qualitative and comparative determinations of
adduct abundance.
[0009] To date, there are no reliable quantitative methods for CEdG
measurement, which is likely due to a lack of suitable isotopically
enriched standards and other barriers to a reliable quantitative
method. Such a method would be a substantial improvement in the
art.
SUMMARY
[0010] In a first embodiment, advanced glycation end products
(AGE), such as N.sup.2-carboxyethyl-2'-deoxyguanosine (CEdG), may
be quantified in a biological sample using liquid chromatography
electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) for
diagnosis, monitoring, and treatment of pathologies involving
metabolic disorders, including abnormal glucose metabolism. Such
pathologies include diabetes and cancer, amongst other metabolic
diseases or disorders. Quantification is achieved by a stable
isotope dilution method using an internal standard. When the AGE is
CEdG, the internal standard is .sup.15N.sub.5-CEdG. The advantage
of having two stereoisomers of CEdG that can be resolved and
quantitated allows for two independent measurements for the same
condition, significantly enhancing the accuracy of the method.
[0011] Detecting physiologically elevated or depressed levels of
AGE in a sample may indicate that the subject from which the sample
was taken has a disease or disorder caused or indicated by such AGE
levels. The quantification method allows for a precise
determination of AGE amounts and thus, allows for sensitive
determination of AGE levels compared to other samples from the same
subject at the same time, other samples from the same subject at
different time points, or other samples from other subjects, such
as a person known not to be affected by a disease. For example,
detecting elevated levels of CEdG in a person indicates
predisposition to or the presence of hyperglycemia or diabetes.
Reaction of double stranded DNA with MG or glucose in vitro
produces primarily N.sup.2-carboxyethyl-2'-deoxyguanosine as a
diastereomeric mixture (FIG. 1). The same type of sample may be
used to compare between various AGE levels, such as a comparison
between AGE levels in a first tissue sample and a second tissue
sample. Alternatively, the AGE levels may be compared between
various types of samples so long as the relative physiological
normal level for each type of sample is known.
[0012] In another embodiment, internal standards for other AGEs are
created using the methods disclosed herein for synthesizing the
internal standard of CEdG. Standards for MS are typically identical
in structure to the intended analyte, but contain stable isotopes
(15N, 13C, 18O) in order to give a different mass to an otherwise
chemically identical substance. The isotope behaves identically to
the intended analyte, has the same retention on chromatography, and
undergoes the same chemistry, and is only distinguishable by
mass.
[0013] In a different embodiment, the quantification methods
described herein may also be used to determine the effectiveness of
a therapy, which may be a test compound or other protocol, intended
to treat or ameliorate an AGE-related disease or disorder (a
"therapeutically effective amount"). Before the therapy is
administered, a first biological sample is taken. After the therapy
has been administered, a second biological sample is taken.
Additional biological samples may also be taken at other time
points during and/or after the therapy. AGE is quantified in the
samples and the difference between AGE levels in the samples is
measured. Other known statistical analysis, such as tests for
statistical significance, may also be applied. If a successful
therapy results in a reduction of the level of AGE and such
reduction is noted after the administration of the therapy, it
indicates that the therapy may be working for its intended purpose.
If AGE levels in the sample are static or increased during the
course of the therapy, it indicates that the therapy may not be
working for its intended purpose of reducing AGE levels. If a
successful therapy results in an increase of AGE levels with a
treatment, the opposite analysis would apply: increases in AGE
levels would indicate the therapy may be working, whereas static or
decreased levels would indicate that the therapy may not be
effective.
[0014] Kits for quantifying AGE levels, such as CEdG levels, are
also contemplated. Such kits facilitate the methods described
herein may contain any of the following: standards such as
.sup.15N.sub.5-CEdG, tubes, labels, reagents such as buffer, and
instructions for use.
[0015] Another embodiment involves measuring urine samples in an
animal model to monitor the dose dependency of LR-90 as it
decreases CEdG levels in vivo.
[0016] Yet another embodiment is measuring the effect of aromatase
inhibitors on CEdG levels, and relatedly, on glycation status. CEdG
levels are measured in a subject undergoing aromatase inhibitory
therapy (AI) to determine the impact of AI on cognitive function
and mental acuity.
[0017] A method of measuring CEdG to predict chemosensitivity of
tumors and to identify cancers that may be treated from targeting
glyoxalase 1 and/or aldose reductase to restore chemosensitivity is
also described. Tumors with elevated levels of CEdG are more
sensitive to chemotherapy. Related methods of inducing production
of CEdG or other AGE products in tumor cells or of administering
CEdG to tumor cells to induce apoptosis and/or increased
sensitivity to chemotherapy are also provided. The effectiveness of
radiotherapy may also be tested by measuring CEdG in tumors.
[0018] A novel synthesis of oligonucleotides containing
site-specifically modified CEdG residues is shown in FIG. 16. Such
synthesis facilitates experiments using CEdG, such as experiments
that investigate the biological consequences of CEdG substitution
in DNA and for serving as internal standards for assays measuring
CEdG.
[0019] These and other embodiments are further explained by
reference to the detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1. The two CEdG diastereomers formed from reaction of
MG with dG.
[0021] FIG. 2. A representative HPLC chromatogram of the reaction
of .sup.15N.sub.5-dG with dl-glyceraldehyde. Peaks A and B
correspond to the two diastereomers of .sup.15N.sub.5-CEdG.
[0022] FIG. 3. Full scan positive ion ESI-MS spectrum for
.sup.15N.sub.5-CEdG diastereomer peak A.
[0023] FIG. 4. Time course product profiles of the reaction of dG
and the A and B stereoisomers of CEdG with 1 M AcOH at 37.degree.
C. The inset shows the HPLC chromatogram of the reaction of CEdG-B
at 450 min.
[0024] FIG. 5. Quantitation of CEdG in normal (light grey) and
diabetic (dark grey) Sprague-Dawley rats. Superscript a (".sup.a"):
Ordinate values represent ad libitum concentrations of the AGE
inhibitor drug LR-90 (mg/L). *P<0.05 and **P<0.01 vs
untreated diabetic animals (Bonferonni's test)(no asterisks).
[0025] FIG. 6. LC-ESI-MS/MS measurements of CEdG diastereomers in
calf thymus DNA subjected to various workup procedures. Hydrolyzed
samples correspond to DNA treated with nuclease P1/alkaline
phosphatase/phosphodiesterase. Calf thymus DNA samples were also
reacted with proteinase K (Extracted) prior to hydrolysis. Levels
of CEdG were measured in the presence or absence of carbonyl
scavenger AG.
[0026] FIG. 7. Reactions of carbonyl scavengers AG and D-P with MG
yield isomeric aminotriazines (top) and 2-acylthiazolidine
(bottom).
[0027] FIG. 8. UV spectra of stock solutions of unlabeled (FIG. 8A)
and isotopically labeled CEdG diasteromers (FIGS. 8B-8C). FIG. 8A:
UV spectra of CEdG(A)(solid line) and CEdG(B)(dotted line) with no
isotopic labeling. Both samples were diluted 1:50;
OD.sub.255(dG)=12,300 OD/M. For CEdG(A), XX-49-A, 28.55 mL; diluted
OD.sub.255=0.450; undiluted OD.sub.255=22.50; conc.=1.83 mM, 52.22
umol @ FW 338.30=17.67 mg. For CEdG(B), XX-49-A, 40.61 mL; diluted
OD.sub.255=0.327; undiluted OD.sub.255=16.35; conc.=1.33 mM, 53.98
umol @ FW 338.30=18.26 mg. FIG. 8B: .sup.15N.sub.5-CEdG(A); 2 uL
stock diluted to 500; OD.sub.255=1.207. FIG. 8C:
.sup.15N.sub.5-CEdG(B); 1 uL stock diluted to 500;
OD.sub.255=0.883.
[0028] FIG. 9. Proton (.sup.1H) NMR of CEdG(A) isomer. The
following parameters apply to the spectrum: transmitter freq:
399.806855 MHz; time domain size: 21340 points; width 5208.33
Hz=13.027115 ppm=0.244064 Hz/pt; number of scans: 512; freq. of 0
ppm: 399.804642 MHz; processed size: 65536 complex points; LB:
0.00; GB: 0.00.
[0029] FIG. 10. Proton (.sup.1H)NMR of CEdG(B) isomer. The
following parameters apply to the spectrum: transmitter freq:
399.806855 MHz; time domain size: 21340 points; width 5208.33
Hz=13.027115 ppm=0.244064 Hz/pt; number of scans: 512; freq. of 0
ppm: 399.804643 MHz; processed size: 65536 complex points; LB:
0.500; GB: 0.00.
[0030] FIG. 11. Carbon data: .sup.13C NMR of CEdG(A). The following
parameters apply to the spectrum: transmitter freq: 100.541493 MHz;
time domain size: 63750 points; width 24509.80 Hz=243.778000
ppm=0.384468 Hz/pt; number of scans: 12000; freq. of 0 ppm:
100.531015 MHz; processed size: 65536 complex points; LB: 0.00; GB:
0.00.
[0031] FIG. 12. Carbon data: .sup.13C NMR of CEdG(B). The following
parameters apply to the spectrum: transmitter freq: 100.541493 MHz;
time domain size: 63750 points; width 24509.80 Hz=243.778000
ppm=0.384468 Hz/pt; number of scans: 27000; freq. of 0 ppm:
100.531015 MHz; processed size: 65536 complex points; LB: 0.500;
GB: 0.00.
[0032] FIG. 13. MS2 and MS3 of sodiated CEdG(A) parent ion obtained
using the Thermo Finnigan LTQ-FT linear ion trap mass spectrometer,
showing the expected molecular fragments for the isotopically
enriched standards.
[0033] FIG. 14. Product ion scans for CEdG(A) and
.sup.15N.sub.5-CEdG(A) at m/z 340 and 345, respectively, showing
the daughter ions at m/z 224 and 229 monitored using a Micromass
Quattro Ultima Triple Quadrupole Mass Spectrometer, showing the
expected molecular fragments for the isotopically enriched
standards.
[0034] FIG. 15. Observed isotopic distributions for
.sup.15N.sub.5-CEdG(A) (FIG. 15A) and .sup.15N.sub.5-CEdG(B) and
the calculated isotopic distribution for
C.sub.13H.sub.17.sup.15N.sub.5NaO.sub.6 (FIG. 15B) The latter was
calculated using the Molecular Weight Calculator, V. 6.38 (FIG.
15C).
[0035] FIG. 16. Synthesis of oligonucleotides containing
site-specifically modified CEdGs.
[0036] FIG. 17. Consistent elevation of CEdG in obese rats, nearly
10-fold in some examples, relative to lean controls. There is
consistently more (S) isomer relative to (R) in biological samples
from both rodents and humans.
[0037] FIG. 18. CEdG levels from tissue-extracted DNA in the liver
(FIG. 18A), pancreas (FIG. 18B) and kidney (FIG. 18C) of Zucker
rats, lean controls and Zucker rats treated with the glycation
inhibitor LR-90.
[0038] FIG. 19. Measurement of urinary CEdG(R) and CEdG(S) isomers
in post-menopausal women undergoing treatment with aromatase
inhibitors.
[0039] FIG. 20. CEdG(R) and CEdG(S) distribution in human solid
tumors and adjacent tissue in lung, breast and kidney cancers.
DETAILED DESCRIPTION
[0040] Quantitative measurement of advanced glycation end products
(AGE) is accomplished using mass spectrometry, such as liquid
chromatography electrospray ionization tandem mass spectrometry
(LC-ESI-MS/MS) and internal standards designed for each targeted
AGE. Such measurements allow for precise determinations of AGE
levels, including small or incremental changes in such levels.
[0041] Diabetes, metabolic disorders, cancer and other diseases may
be diagnosed by measuring N.sup.2-carboxyethyl-2'-deoxyguanosine
(CEdG) levels alone or in conjunction with other AGE levels in
biological samples. CEdG levels are measured using liquid
chromatography electrospray ionization tandem mass spectrometry or
other reliable means. The CEdG levels from the sample are then
compared to physiologically normal CEdG levels. Methods for further
determining the efficacy of therapies or treatments applied to
those disorders comprise measuring the effect the putative
therapeutic have on the CEdG levels in an individual receiving it.
The subject having its AGE levels and/or the efficacy of treatment
measured is preferably a mammal, such as a human.
[0042] Thus, one method of quantifying one or more advanced
glycation end products in a sample, comprises obtaining a
biological sample from a subject; and performing liquid
chromatography electrospray ionizing tandem mass spectrometry assay
on the sample using a stable isotope dilution and an internal
standard to determine how much AGE is in the sample. When the AGE
is N.sup.2-carboxyethyl-2'-deoxyguanosine (CEdG), the internal
standard is .sup.15N.sub.5-CEdG. With CEdG quantities in hand,
abnormal levels may indicate metabolic disorders, cancers, or
diabetes. Upon detecting the levels, efficacies of various
treatments may be determined using AGE levels as a marker for the
success of the treatment.
[0043] Metabolic diseases cover a wide range of disorders including
carbohydrate metabolism, amino acid metabolism, organic acid
metabolism, mitochondrial metabolism, porphyrin metabolism, fatty
acid oxidation disorders, purine and pyrimidine metabolism, steroid
metabolism, mitochondrial metabolism, peroxisomal and lysosomal
storage disorders, and glycolytic metabolic disorders, such as
glyolytic cancers. A glycolytic cancer is a cancer that is caused
or influenced by abnormal sugar processing, such as with glycation.
Conditions which result in the impairment of glucose regulation
such as diabetes and metabolic syndrome have been shown to
significantly increase the risk for cancers of the breast, liver,
pancreas, colon, cervix and endometrium. In the case of
hyperglycemia and/or diabetes, an elevated level of CEdG, as
compared to normal physiological levels of CEdG, indicates that the
subject has diabetes.
[0044] A sensitive LC-ESI-MS/MS method for the measurement of CEdG
in urine or double-stranded DNA is used. Quantification is achieved
by the stable isotope dilution method using synthetic
.sup.15N.sub.5-CEdG as an internal standard. Urinary CEdG was
measured in normal and streptozoticin-induced diabetic rats, and it
was shown that adduct levels are significantly increased following
the onset of hyperglycemia. LC-ESI-MS/MS was used to demonstrate a
dose-dependent reduction in CEdG in response to administration of
LR-90, an inhibitor of AGE formation. Measurement of CEdG from
hydrolyzed and dephosphorylated double-stranded DNA was complicated
by the fact that MG was present during the enzymatic workup. This
was found to react with DNA during sample workup leading to
artifactual overestimation of CEdG levels. In order to circumvent
this problem, adventitious MG was sequestered by the addition of
carbonyl scavengers such as aminoguanidine (AG) and D-penicillamine
(D-P) prior to workup, resulting in stable and reproducible
determinations. In the case of glycolytic cancers, such as breast
cancer, a reduced level of CEdG, as compared to normal
physiological levels of CEdG, indicates that the subject has
cancer.
[0045] Materials and instrumentation.
.sup.15N.sub.5-2'-deoxyguanosine was purchased from Silantes
(Munich, Germany, lot # dG-N-0507-1/2); DL-glyceraldehyde (95%),
calf thymus DNA was from Sigma (St. Louis, Mo.), and ammonium
acetate (1M, pH 7 solution) from Fluka (Buchs, Switzerland).
Phosphate salts were A.C.S. reagent grade from J. T. Baker
(Phillipsburg, N.J.). High performance liquid chromatography (HPLC)
grade CH.sub.3CN was purchased from Fisher Scientific (Fair Lawn,
N.J.). All water was purified to a resistivity of 18.2 M.OMEGA.
using a Nanopure Diamond system by Barnstead International
(Dubuque, Iowa). Solid phase extractions were performed using 1 ml
strata-X-C cation mixed mode cartridges (Phenomenex, Torrance
Calif.). Nuclease P1 was purchased from US Biologicals (Swampscott,
Mass.). Phosphodiesterase II from bovine spleen and alkaline
phosphatase from bovine intestinal mucosa was purchased from
Sigma-Aldrich. HPLC separations were performed using a
Hewlett-Packard Series 1100 Liquid Chromatography system equipped
with a diode-array detector. Ultraviolet spectra were collected on
an Ultrospec 3000 pro (Amersham Biosciences, Piscataway, N.J.).
Mass analysis of synthetic .sup.15N.sub.5-CEdG was performed using
a Thermo Finnigan LTQ-FT linear ion trap mass spectrometer (San
Jose, Calif.) in the Mass Spectrometry-Proteomics Core Facility of
the City of Hope.
[0046] LC-MS/MS analyses of CEdG in biological samples were carried
out using a Micromass Quattro Ultima Triple Quadrupole Mass
Spectrometer (Beverly, Mass.) interfaced to an Agilent 1100
Capillary HPLC system (Palo Alto, Calif.) equipped with a Synergi
C.sub.18 analytical column (4.mu., 150.times.2.0 mm; Phenomenex,
Torrance, Calif.). .sup.1H NMR spectra were recorded at 400 MHz on
a VNMRS spectrometer (Varian, Inc., Palo Alto, Calif.) in the
Synthesis and Biopolymer Core Facility of the City of Hope. 1D and
2D NMR data was processed using the Spinworks shareware program
(version 2.5.5), copyright 1999-2006 by Kirk Marat and available
from the University of Manitoba website.
[0047] Synthesis and characterization of .sup.15N.sub.5-CEdG.
DL-Glyceraldehyde was used to generate methylgloxal (MG) in situ
via guanine catalyzed dehydration..sup.17 .mu.L-Glyceraldehyde (9.5
mg) was added to 10 mg of .sup.15N.sub.5-labeled dG, 12.3 mg
potassium dihydrogen phosphate, and 24.0 mg disodium hydrogen
phosphate in 87.7 .mu.L H.sub.2O. The heterogeneous reaction
mixture was vortexed and placed in a heat block at 40.degree. C.
Reactions were worked up following complete dissolution of solids
(.about.14-17 days) yielding a yellow-red viscous solution.
Products were purified by HPLC in 10-15 .mu.L aliquots on a
10.times.50 mm Waters XTerra MS C.sub.18 2.5.mu. column using a
(Et).sub.3NH.sub.4OAc (50 mM, pH 7)/CH.sub.3CN gradient. The
CH.sub.3CN concentration was raised from 0 to 4.0% in the first 5
minutes, from 4.0 to 6.5% over 30 minutes; held at 6.5% for 5
minutes, then raised to 90% to wash residual material off the
column. Diastereomers CEdG-A and B eluted at 24 and 29 minutes,
respectively (FIG. 2).
[0048] Fractions were lyophilized to dryness prior to resuspension
in 18.2 M.OMEGA. H.sub.2O. Concentrations of stock solutions were
calculated by UV using a molar extinction coefficient of 12,300 @
255 nm. See, for example, FIG. 8. Mass analyses of
.sup.15N.sub.5-CEdG diastereomers were conducted using a
Thermo-Finnigan LTQ FT ion trap mass spectrometer in the positive
ion mode. A full scan MS for CEdG-A is shown in FIG. 3. The most
intense ion was observed for the sodiated peak,
C.sub.13H.sub.17.sup.15N.sub.5NaO.sub.6.sup.+: m/z 367.18 (obs),
m/z 367.09 (calc). .sup.1H NMR assignments for CEdG-A: .sup.1H NMR
(400 MHz, d.sub.6-DMSO, 18.degree. C.) .delta.10.60 (s, 1H, N1-H),
.delta. 7.93 (s, 1H, C8-H), .delta. 6.76 (d, 1H, C2-NH), .delta.
6.12 (dd, 1H, C1'-H), .delta. 5.30 (d, 1H, C3'-OH), .delta. 4.89
(vbr, 1H, C5'-OH), .delta. 4.36 (m, 1H, C2-NH--CH), .delta. 4.32
(m, 1H, C4'-H), .delta. 3.81 (m, 1H, C3'-H), .delta. 3.50 (ddd, 2H,
C5'-H.sub.2), .delta. 2.64 (ddd, 1H, C2'-H), .delta. 2.18 (ddd, 1H,
C2'-H), .delta. 1.39 (d, 3H, C2-NH--CH--CH.sub.3). .sup.13C NMR
assignments for CEdG-A: (100.5 MHz, d.sub.6-DMSO, 18.degree. C.)
.delta. 174.1 (C2-NH--CH--COOH), .delta. 156.3 (C6), .delta. 151.5
(C2), .delta. 149.9 (C4), .delta. 136.1 (C8), .delta. 117.1 (C5),
.delta. 87.6 (C3'), .delta. 82.9 (C1'), .delta. 70.8 (C4'), .delta.
61.7 (C5'), .delta. 49.0 (C2-NH--CH), .delta. .about.39.5 (C2'),
.delta. 17.7 (C2-NH--CH--CH.sub.3). .sup.1H and .sup.13C NMR
assignments for CEdG-B are nearly identical to the A isomer.
[0049] Synthesis of oligonucleotides containing site-specifically
modified CEdG residues. A synthetic scheme was devised for the
quantitative preparation of oligonucleotides containing CEdG that
can be readily accommodated on any standard DNA synthesizer using
the conventional phosphoramidite technology. Oligos containing only
pure D or L CEdG were prepared in a stereochemically pure manner
using D or L alanine in a reaction that proceeds with retention of
configuration. A NPE protected 2-fluoropurine phosphoramidite
derivative was introduced into the polymer during standard
oligonucleotide synthesis, and the reaction with D or L alanine was
carried out prior to any deprotection step.
[0050] Specifically, stereochemically pure (R) or (S) CEdG
oligonucleotides were synthesized by nucleophilic substitution with
either (R) or (S) alanine on 2-fluorodeoxyinosine (2-FdI)
containing oligos followed by deprotection and purification.
Oligonucleotides were prepared using an ABI394 DNA synthesizer
loaded with either standard or 2-F-dI-CE phosphoramidites (0.2
.mu.M scale). For the preparation of CEdG containing
oligonucleotides, F-dI-containing fully-protected oligomers still
bound to the CPG support were suspended in an aqueous solution of
1M D- or L-alanine in 250 mM potassium carbonate at 50.degree. C.
for 40 hours. Complete removal of all protecting groups was
achieved by extended reaction at 50.degree. C. in concentrated
ammonia for 7 days. Separation of the desired oligonucleotide from
failure sequences and other impurities was achieved by ion-pairing
chromatography on a 10 mm.times.250 mm X Bridge Prep C18 5 .mu.m
column (Waters, Milford, Mass.), using a 40 minute 9.0% to 9.5%
gradient of acetonitrile vs 100 mM triethylammonium acetate (TEAA,
Fluka, Milwaukee, Wis.) at a constant 45oC. All oligonucleotides
were characterized by rechromatography under the indicated
conditions and analyzed by ESI-FT/MS on an LTQ-FT (Thermo-Finnigan,
San Jose, Calif.) in the Mass Spectrometry Core of the City of Hope
Cancer Center.
[0051] This new synthesis is superior to previously known syntheses
for CEdG because it allows for the preparation of oligos containing
stereochemically pure (R) or (S) CEdG in high yield. Oligos
containing uniquely substituted CEdG residues are used to calibrate
the biological measurement of CEdG by serving as internal
standards. They are also used in biochemical assays for examining
the biological consequences of site-specific CEdG substitution in
DNA, including, but not limited to, aspects of their repair and
mutagenic potential (FIG. 16). This synthetic scheme may also be
used to make site specific substitutions for other AGEs.
[0052] Stable Isotope Dilution. Internal standards for other AGEs
are usually contain stable isotopes (15N, 13C, 18O) to create a
different pass from the related analyte. Different concentrations
of the stable isotope substituted compounds are prepared and
analyzed by MS in order to determine the response height of the ion
current as a function of different concentrations. A calibration
plot is made of concentration vs ion current response. This is
typically a linear plot of concentrations ranging from anticipated
lowest detectable amounts to highest expected. The ion current
response increases with concentration. To measure CEdG in a
biological sample, a known amount of stable isotope standard is
"spiked" into the sample. Since the CEdG in the sample and the CEdG
standard have different molecular weights, they can be resolved by
MS. The ion current response of the CEdG in the sample is compared
to the response of the spiked istopically enriched CEdG. Since the
concentration of isotopically enriched standard in the sample is
known, comparison allows for calculation of the amount of CEdG in
the biological sample by fitting to the calibration plot.
[0053] Stability studies of CEdG in acidic solution. A 1.25 mM
solution of CEdG-A, B or dG in 100 .mu.L of 1M AcOH (pH 2.4) was
stirred at 37.degree. C. 10 .mu.l aliquots were removed
periodically and added to 40 .mu.L of 2M TEAA (pH 7.0). HPLC
product analyses were performed using an Alltech HS HyperPrep 100
BDS C18 8.mu. column. A gradient of 0 to 4% CH.sub.3CN over 5 min
was followed by 6.5% CH.sub.3CN over 30 min. TEAA (pH 7) was kept
constant at 50 mM. The ratio of free base (CEG or G) to intact
nucleoside (CEdG or dG) was calculated by integration of the
corresponding HPLC peaks (see inset in FIG. 4). The CEG free base
was identified as Peak A by ESI-MS in the negative ion mode.
C.sub.8H.sub.8O.sub.3N.sub.5, observed: m/z 222.064; calculated:
m/z 222.063.
[0054] Animal Studies. All animal studies were carried out in
compliance with the policies outlined in NIH Publication No. 85-23
"Guide for the Care and Use of Laboratory Animals." Male
Sprague-Dawley rats were rendered diabetic by injection of
streptozoticin and maintained as previously described..sup.18 The
AGE inhibitor LR-90 was administered ad libitum at concentrations
ranging from 2.5-50 mg/L. Rats were housed in metabolic cages and
urine was collected over a 24 hour period with several drops of
toluene to inhibit microbial growth. Urine samples were stored at
-80.degree. C. prior to LC-MS/MS analysis for CEdG. The data in
FIG. 5 represent 3 replicates from n different animals:
non-diabetic controls, n=6; non-diabetic treated with 50 mg/L
LR-90, n=5; diabetic control, n=3. For diabetic rats treated with
varying doses of LR-90: 2.5 mg/L, n=4; 10 mg/L, n=5; 25 mg/L, n=6;
50 mg/L, n=8.
[0055] Urine sample preparation. CEdG was concentrated from urine
by solid phase extraction. A 1 ml strata-X-C cartridge was
pre-conditioned by the sequential addition of 1 ml MeOH/CH.sub.3CN
(1:1) followed by 2.times.1 ml 2% H.sub.3PO.sub.4. Then
.sup.15N.sub.5-CEdG was added as an internal standard (final
concentration 5 .mu.g/ml), the sample was acidified with 10 .mu.l
of 86% H.sub.3PO.sub.4, and finally 0.4 mL of urine was introduced
via suction filtration. The cartridge was then washed with
sequential additions of 1 ml 0.1% H.sub.3PO.sub.4 and 1 ml MeOH and
then dried under vacuum for 1 minute. Finally, CEdG and
.sup.15N.sub.5-CEdG containing fractions were eluted from the
cartridge with 1 mL 3% NH.sub.4OH in MeOH:CH.sub.3CN (2:8 v/v). The
eluent was evaporated to dryness in a centrifugal concentrator and
reconstituted with 200 .mu.l 0.1% formic acid prior to LC-MS/MS
injection.
[0056] Preparation of Mononucleosides from DNA. Calf Thymus or
Tissue-extracted DNA (100 .mu.g) was dissolved in 80 .mu.L of
autoclaved 18.2 M.OMEGA.H.sub.2O containing 20 .mu.L of sodium
acetate (100 mM, pH 5.5), 20 .mu.L of 1.times.TBE, 1.5 .mu.L of 50
mM ZnCl.sub.2, and 2.37 .mu.L of a 100 mM AG or D-P stock solution.
DNA was denatured at 95.degree. C. for 5 min on a PCR heating block
and then brought to 4.degree. C. for 5 min. After equilibration to
45.degree. C., 1.5 .mu.L of 10 U/.mu.L nuclease P1 was added.
Alkaline phosphatase (4 .mu.L of 8 U/.mu.L), 1 U of bovine
phosphodiesterase, and 14 .mu.L of 100 mM CaCl.sub.2 were added
after 1 hour, and the hydrolysis/dephosphorylation was continued
for another 7 hours. DNA concentrations were determined by UV
spectroscopy (1 OD.sub.260=50 .mu.g/ml) and samples were stored at
-80.degree. C. prior to MS analyses. A 5 .mu.L aliquot of digest
was diluted to 200 .mu.L and used for quantitation of
2-deoxyguanosine by HPLC integration using a Beckman C-18 reverse
phase (25 cm.times.4.6 mm) column (Fullerton, Calif.). Separation
was achieved isocratically using a mobile phase of 6% MeOH, 0.1%
acetic acid in water.
[0057] DNA isolation from human tissue. Breast tumor and adjacent
normal tissue was obtained from the frozen tumor bank of the City
of Hope Pathology Core. A pea-sized section (.about.100 mg) of
tissue was minced and suspended in 1.2 mL of digestion buffer (100
mM NaCl, 10 mM Tris HCl, pH 8, 25 mM EDTA, pH 8, 0.5% SDS, 0.2
mg/mL proteinase K, 10 mM D-penicillamine) and incubated at
50.degree. C. in a water bath for 12-18 h. DNA was then extracted
using an equivalent volume of phenol/chloroform/isoamyl alcohol
(25:24:1). The aqueous fraction was separated and 0.5 volumes of
ammonium acetate and 2 volumes of 100% ethanol were added. The DNA
was spooled, washed twice with 70% ethanol, pelleted, and
resuspended in autoclaved 18.2 MS.OMEGA. water. The enzymatic
hydrolysis was carried out as described above.
[0058] LC-ESI-MS/MS. Quantification of CEdG was performed using a
LC-MS/MS method. Measurement of 8-oxo-dG was performed as
previously described..sup.19 CEdG and .sup.15N.sub.5-CEdG (internal
standard) were synthesized and purified. Measurements were
performed using an Agilent 1100 Capillary LC system (Agilent
Technologies, Palo Alto, Calif.) in line with a Micromass Quattro
Ultima Triple Quadrupole Mass Spectrometer (Micromass, Beverly,
Mass.) operating in positive-ion mode. The detector settings were
as follows: capillary voltage, 3.5 kV; cone voltage, 18 V;
collision cell voltage, 11 V; source temperature, 350.degree. C.;
desolvation temperature, 150.degree. C.; cone gas flow, 620
liter/h; and desolvation gas flow, 500 liter/h. The mass
transitions monitored for CEdG and .sup.15N.sub.5-CEdG were
340.3.fwdarw.224.3 and 345.4.fwdarw.229.4 respectively. HPLC was
accomplished using isocratic conditions with a mobile phase of 15%
aqueous MeOH with 0.1% formic acid on a Prodigy ODS C-18 (25
cm.times.2.0 mm.times.5 micron) column (Phenomenex, Torrance,
Calif.). The flow rate was 0.2 ml/min with a total run time of 30
min. The retention times for CEdG diastereomers A and B using these
conditions were 9.3 and 16 min, respectively. The lower limit of
quantitation for CEdG, defined as a peak height of .gtoreq.5.times.
baseline noise, was 0.1 ng/ml in the starting solution or 0.2 pg on
column.
[0059] For urine analyses and calf thymus DNA digests, calibration
curves were constructed using 0.75, 1.5, 3, 6, 12, 24, and 48 ng/mL
of synthetic CEdG in urine or in blank nucleoside digestion buffer.
For human breast tissues, CEdG concentrations used for calibration
were 0.19, 0.38, 0.75, 1.5, 3, and 6 ng/mL. Linearity of the
calibration curves were demonstrated with R-squared values of
.gtoreq.0.996. Inter- and intra-day accuracy of the assay across
the range of the standard curve was established to be 96% and 94%
of target concentrations, respectively. The assay was also
determined to be unbiased with both inter- and intra-day precision
within +6%. Quantification of 2'-deoxyguanosine (dG) was performed
by HPLC integration of DNA digests and final values were expressed
as CEdG/10.sup.7dG.
[0060] Urine extracts or mononucleoside digests were spiked with 20
.mu.L of 100 ng/mL .sup.15N.sub.5-labeled CEdG and 10 .mu.L of 86%
phosphoric acid. Samples were then loaded onto strata-X-C cation
mixed mode columns that had been pre-conditioned with
MeOH/CH.sub.3CN (1:4) followed by 2% phosphoric acid. After sample
loading, columns were washed with 0.1% phosphoric acid, followed by
MeOH. Nucleosides were eluted with 3% ammonium hydroxide in
MeOH/CH.sub.3CN (1:4) and evaporated to dryness in a centrifugal
concentrator. Samples were reconstituted with 100 .mu.L of 0.1%
formic acid and analyzed directly by LC-MS/MS. Recovery of CEdG
diastereomers and .sup.15N.sub.5-CEdG from urine and mononucleoside
digests was determined to be 85+/-0.9%.
[0061] Synthesis and characterization of CEdG isotopomers.
Isotopomers of CEdG were prepared by a modification of the method
of Ochs and Severin..sup.17 Reaction of .sup.15N.sub.5-dG with
(dl)-glyceraldehyde in phosphate buffer afforded the desired
products as a .about.1:1 mixture of diastereomers in .about.60%
yield. Unenriched CEdG diasteromers were prepared in an analogous
manner. The N.sup.2 amino group of dG catalyzes the dehydration of
glyceraldehyde to yield the hemiacetal of MG in situ, which then
reacts to provide CEdG either directly by condensation at N.sup.2
or alternatively via the rearrangement of an intermediate N.sup.1,
N.sup.2 cyclic diol. The two diastereomers of CEdG were readily
resolved by HPLC and eluted at 24 and 29 minutes (FIG. 2) on a
C.sub.18 reverse phase column. In spite of significant differences
in chromatographic retention times, both the proton and carbon NMR
spectra for CEdG-A and B were essentially superimposable, with the
chemical shift differential on the order of <0.1 ppm for proton
and <1.0 ppm for carbon.
[0062] Mass analyses of the CEdG isotopomers were performed using a
Thermo Finnigan LTQ ion trap mass spectrometer in the positive ion
mode. The most intense signal in the parent ion spectrum of the
isotopically enriched standard corresponded to the sodium salt of
.sup.15N.sub.5-CEdG at m/z 367 [PNaH].sup.+ (FIG. 3). The disodium
salt [PNa.sub.2].sup.+ and the dihydro adduct [PH.sub.2].sup.+ were
also observed at m/z 389 and 345, respectively. Collision induced
dissociation of the m/z 367 parent ion gave rise primarily to the
sodiated base ion [BNaH].sup.+ at m/z 251. The observed isotopic
distribution for C.sub.13H.sub.17.sup.15N.sub.5NaO.sub.6 was found
to be in good agreement with the calculated values.
[0063] Stability of CEdG to acid-catalyzed depurination and
sidechain isomerization. The chemical stability of CEdG was
examined as an important criterion for evaluating its suitability
as a quantitative biomarker. Purified stereoisomers of synthetic
CEdG were subjected to acidic conditions (1 M AcOH at 37.degree.
C.) and the extent of released free base and diastereomer
interconversion was monitored by HPLC as a function of time.
Analogous experiments were performed for dG and the results are
presented in FIG. 4. The approximate half-lives for depurination
were 750 and 500 min for the A and .beta. isomers respectively,
whereas dG was observed to be less stable, with a half-life of 440
min under these conditions. No racemization of the sidechain
stereocenter was detected during acidic hydrolysis, i.e., no
interconversion of CEdG isomers A and B was observed.
[0064] Urinary CEdG measurement in diabetic rats. A diabetic animal
model was used to examine the relationship between glycemic status
and CEdG levels. Rats rendered diabetic by streptozoticin (STZ)
treatment possess elevated MG relative to normal controls and thus
appeared likely to exhibit an increased burden of CEdG adducts. The
effect of AGE inhibitor, LR-90, was also examined. The results of
these experiments are shown in FIG. 5. Analyses of urine from
non-diabetic control animals collected over a 24 hr period revealed
mean CEdG levels of 77 pg/ml (FIG. 5). The induction of diabetes
increased the level of excreted CEdG by .about.4 fold.
Administration of LR-90 to diabetic rats ad libitum at a dose
corresponding to 2.5 mg/L resulted in a 2.3 fold decrease in CEdG
titer. Increasing concentrations of LR-90 led to a dose dependent
reduction in CEdG, and at 25 mg/L the adduct level in urine was
comparable to that of non-diabetic animals. In contrast,
administration of LR-90 at doses up to 50 mg/L in normal controls
had no significant effect on CEdG levels. 8-oxo-dG was also
measured as an indicator of oxidative stress in normal and diabetic
rats; however, excreted 8-oxo-dG in diabetic animals was not
statistically different (P>0.05) from controls.
[0065] CEdG in organs of Zucker fatty rats. The Zucker rat is a
morbidly obese, hyperinsulinemic model for Type 2 diabetes
resulting from homozygous knockout of the leptin receptor. In order
to determine whether elevated circulating glucose in the Zucker rat
correlates with increased tissue DNA glycation, CEdG levels from
tissue-extracted DNA were measured in selected organs and compared
to lean controls and to Zucker rats treated with the glycation
inhibitor LR-90. Data for liver, pancreas and kidney are shown in
FIG. 18. Relative to lean rats, CEdG levels were found to be
elevated only in kidneys. In lean animals, CEdG was below the level
of detection in 9/9 animals, whereas it was elevated in 5/9 Zucker
rats. All three organs of Zucker rats had a net lowering of CEdG
levels following treatment with LR-90. These data show that CEdG
determination can be used to monitor tissue glycation levels in
response to chemotherapy.
[0066] CEdG in calf thymus DNA. Commercial grade calf thymus DNA
was used as a model substrate for developing a protocol for CEdG
quantitation in double-stranded DNA. DNA was hydrolyzed and
dephosphorylated by sequential addition of nuclease P1, alkaline
phosphatase and phosphodiesterase. Then, mononucleosides were
concentrated by solid phase extraction prior to LC-MS/MS analyses.
The results of these experiments are shown in FIG. 6. Initial
determinations yielded values of CEdG in the range of 60-66
CEdG/10.sup.6 dG. These surprisingly high levels showed that some
CEdG may have been formed artifactually during the hydrolysis and
dephosphorylation. Additional CEdG may have been formed due to the
release of MG from the protein reagents used in the workup during
prolonged incubation. Proteins can bind MG reversibly, and up to
90% of cellular MG may be sequestered in this manner. In order to
prevent additional reactions of adventitiously generated MG with
DNA, carbonyl scavenging agents AG or D-P were added prior to DNA
digestion and dephosphorylation. These reagents sequester MG and
other alpha-oxoaldehydes by forming stable cyclic aminotriazine and
thiazolidine derivatives respectively. Concentrations of AG from
0.5 to 50 mM were added prior to workup, and CEdG levels were
measured in order to determine the optimal concentration required
to achieve stable, reproducible levels. The addition of 10 mM AG
prior to sample processing resulted in a modest but significant
drop in adduct levels (45-50 CEdG/10.sup.6 guanines) in calf thymus
DNA, suggesting that .about.15 CEdG/10.sup.6 guanines were formed
as a direct result of the hydrolysis and dephosphorylation
protocol.
[0067] Since the extraction of DNA from biological samples requires
extended reaction with proteinase K (up to 24 h), it was
investigated whether this treatment could also contribute to
artifactual CEdG formation. Accordingly, calf thymus DNA was
subjected to mock proteolysis prior to hydrolysis and workup in the
absence of carbonyl scavenger. FIG. 6 reveals an increase in adduct
levels significantly higher than those observed following
hydrolysis alone, with values ranging from 80-100 CEdG/10.sup.6
guanines. The addition of 10 mM AG in two aliquots prior to the
mock lysis treatment and hydrolysis/dephosphorylation steps
resulted in a drop in measured CEdG levels comparable to that
observed previously for calf thymus DNA subjected only to the
hydrolysis/dephosphorylation in the presence of AG. No apparent
stereoisomer bias was detected in any of these samples, i.e., the
ratio of CEdGA:CEdGB was not significantly different from 1:1.
[0068] Measurement of urinary CEdG in post-menopausal women
undergoing treatment with aromatase inhibitors. One noted
side-effect of treatment with aromatase inhibitors (AI) in cancer
therapy is an impairment of cognitive function, which may be linked
to enhanced glycation in the brain. Enhanced brain glycation is a
contributing factor in the pathology of Alzheimer's disease. In
order to examine whether treatment with aromatase inhibitors can
affect glycation status, urine from 32 patients was collected just
prior to and 6 months following administration of AI, and levels of
CEdG were measured in urine. Data for the (R) and (S) isomers of
CEdG are shown in panels A and B, respectively, of FIG. 19. In the
case of the (R) isomer, 12/32 patients show significantly higher
levels after AI treatment, a trend also observed for 14/32 patients
when levels of the (S) isomer are considered. Some of these
post-treatment levels are very high, much higher than any observed
pre-treatment levels. There is also good consistency between the
two independent biomarkers. For example, in patients 3, 6, 9, 12,
13, 17, 18, 20, 23, 24, 29 and 30, both stereoisomers are elevated
post-AI treatment. If these changes are correlated with decreased
mental acuity over time, CEdG measurement can also be used to
identify patients at risk for cognitive impairment. Additionally,
one or more CEdG inhibitors, such as LR-90, may be administered to
a subject undergoing chemotherapy in order to prevent or reduce the
cognitive impairment that may accompany chemotherapy.
[0069] CEdG measurement in human solid tumors vs adjacent tissue.
Frozen tumor specimens and adjacent tissue were obtained from the
City of Hope Tumor Bank. DNA was extracted as described and
analyzed for CEdG. Results are shown in FIG. 20 for (R) and (S)
CEdG in lung, breast and kidney cancers. In lung cancers CEdG was
observed at lower levels in tumor relative to adjacent tissue in
the majority of samples. This same phenomenon was observed for the
single breast cancer sample analyzed. These trends are followed for
both isomers. In the case of kidney cancers, the situation is more
complex, with samples 3 and 6 showing the opposite trend of higher
CEdG in tumor relative to adjacent tissue. In sample 6, the levels
of (R) and (S) isomers were 13 and 18 fold higher respectively in
tumor relative to adjacent tissue. Other samples, such as 1, 4 and
5, follow the trend observed in the lung and breast samples.
[0070] These variations in CEdG between tumor and adjacent tissue
represent the corresponding levels of glycolytic stress. In order
to avoid the pro-apoptotic effects of methylglyoxal produced as a
result of enhanced glycolysis, solid tumors must restrict its
accumulation. Tumors with lower levels of CEdG relative to adjacent
tissue, can successfully minimize their glycolytic stress in spite
of maintaining elevated glycolysis. This is likely due to
overexpression of the methylglyoxal scavenging enzymes glyoxalase 1
and aldose reductase in tumors, as well as enhanced removal of CEdG
from DNA by repair enzymes. Tumors with elevated levels of CEdG
relative to adjacent tissue are predicted to be genetically
unstable, and more sensitive to chemotherapy as a result of the
cytotoxic accumulation of methylglyoxal. Thus, another embodiment
is a method of predicting which tumors of a cancer patient are more
susceptible to chemotherapy by testing CEdG levels in tumor
samples. If the CEdG levels are high, then the tumor is more likely
to be receptive to chemotherapy treatment. Measurement of CEdG can
also be used to identify which cancers which can benefit from
targeting glyoxalase 1 and/or aldose reductase, in order to restore
their sensitivity to chemotherapy. CEdG measurement can provide a
direct means for identifying tumors most likely to benefit from
these approaches.
[0071] Quantitation of CEdG in a human breast tumor and adjacent
normal tissue. Many cancer cells in the hyopoxic tumor
microenvironment primarily utilize glycolysis to meet their
energetic demands. This glycolytic phenotype (Warburg effect) is
characterized by constitutive cell surface expression of glucose
transporter proteins such as GLUT-1, and forms the basis for the
diagnostic use of .sup.18FDG-PET in the imaging of breast and other
cancers..sup.26,27 Enhanced glyocolytic flux suggests that breast
tumors might exhibit abnormal levels of AGEs including CEdG.
Accordingly the levels of CEdG diastereomers were measured in DNA
extracted from a clinical breast tumor specimen as well as adjacent
normal tissue. The data in Table 1 reveal some significant
(P<0.05) differences in the levels of CEdG between tumor and
normal tissue. Both stereoisomers were observed at .about.3-fold
higher levels in normal relative to tumor tissue (CEdG-A, P=0.02;
CEdG-B, P=0.003). In the column under CEdG/107dG, "a" indicates
P=0.08 versus CEdG-B in normal issue; "b" indicates P=0.02 versus
CEdG-A in adjacent normal tissue; "c" indicates P=0.003 versus
CEdG-B in adjacent tumor tissue; and "d" indicates P=0.03 versus
CEdG-A in tumor tissue.
TABLE-US-00001 TABLE 1 CEdG isomers from a human breast tumor and
adjacent normal tissue. CEdG (fmol) dG (fmol) CEdG/10.sup.7 dG
CEdG-A Normal 234 .+-. 24.9 1.91 .times. 10.sup.8 12.3.sup.a .+-.
1.3 Tumor 247 .+-. 11.6 6.48 .times. 10.sup.8 3.9.sup.b .+-. 0.2
CEdG-B Normal 151 .+-. 4.98 1.91 .times. 10.sup.8 7.9.sup.c .+-.
0.3 Tumor 173 .+-. 6.64 6.48 .times. 10.sup.8 2.7.sup.d .+-. 0.1
.sup.aP = 0.08 versus CEdG-B in normal tissue. .sup.bP = 0.02
versus CEdG-A in adjacent normal tissue. .sup.cP = 0.003 versus
CEdG-B in adjacent tumor tissue. .sup.dP = 0.03 versus CEdG-A in
tumor tissue.
[0072] Within normal tissue, the levels of CEdG-A and B were not
significantly different (P=0.08), while in tumor there was a small
bias favoring CEdG-A (P=0.03). Levels of CEdG in DNA extracted from
either breast tumor or adjacent tissue in the absence of carbonyl
scavenger were .about.1.5-2.0 fold higher; however, artifactual
formation was inhibited by the addition of 10 mM D-penicillamine in
two aliquots during both the cell lysis/DNA isolation and
hydrolysis/dephosphorylation steps. .sup.15N-enriched isotopomers
of CEdG differing from the unlabelled adducts by 5 amu were
synthesized, which provided sufficient mass resolution for accurate
and reproducible quantitation using the stable isotope dilution
method.
[0073] The ability to simultaneously resolve and quantitate both
diastereomers of CEdG provides two independent parameters for
assessing DNA glycation levels within a single sample. The
biological significance of the CEdG diastereomer ratio in vivo may
reflect stereochemical biases in adduct repair or polymerase
bypass. Of course, examination of the CEdG stereoisomer
distribution in vivo by LC-ESI-MS/MS would only be meaningful if
the rate of stereochemical interconversion was negligible.
Regarding overall adduct stability, loss of the CEG base from
either stereoisomer during workup would result in the generation of
abasic sites leading to an underestimation of true nucleoside
adduct levels, which was of particular concern since CEdG undergoes
depurination more readily than dG at elevated temperatures. The
extent of depurination and racemization was quantified by
monitoring free base formation and isomer interconversion under
acidic conditions at 37.degree. C. rather than at non-physiological
temperatures. FIG. 4 shows that the CEdG diastereomers possess
similar stability, and are slightly more resistant to depurination
under acidic conditions than dG. This fact, together with the
prohibitive barrier to stereochemical interconversion, indicates
that determination of CEdG diastereomer ratios may be plausibly
used in quantitative biomarker studies. Various quantifications of
CEdG are found in FIGS. 9-15.
[0074] One important confounding factor in the quantitation of
adducts resulting from oxidative or oxoaldehyde DNA modification is
artifactual product formation during sample isolation and workup.
The problems surrounding the measurement of 8-oxo-dG using GC-MS
and/or mildly oxidizing workup conditions have been detailed
previously..sup.36-38 In the case of CEdG adducts, the presence of
MG during the workup could complicate the accurate determination of
endogenous levels. The effects of carbonyl scavenger addition prior
to the enzymatic digestions were examined due to the high
background levels of CEdG detected in reagent grade calf thymus
DNA. Scavengers such as AG and D-P react rapidly with MG and other
oxoaldehydes to yield aminotriazines and thiazolidines respectively
(FIG. 7) which are relatively unreactive electrophiles.
D-penicillamine reacts with MG 60 times faster than AG, and thus
may be more advantageous for CEdG determinations requiring DNA
isolation from complex tissue matrices.
[0075] MG bound reversibly to proteins was predominantly
responsible for the formation of DNA glycation artifacts observed
during the isolation and workup of dsDNA. Extraction and workup
procedures which expose DNA for extended periods to cell lysates
and partially purified enzyme reagents increase the probability for
the ex vivo formation of CEdG, necessitating the need for carbonyl
scavengers. MG-BSA conjugates prepared by incubating MG with BSA
can be used as reagents to induce DNA damage in cultured mammalian
cells. The data in FIG. 6 suggest that the addition of AG or D-P
can largely eliminate artifactual CEdG formation. Minimizing
exposure to proteins by shortening the enzymatic lysis and
hydrolysis/dephosphorylation steps may also reduce the requirement
for carbonyl scavengers.
[0076] A diverse array of tumor and corresponding control tissues
are examined in order to determine whether the trends noted in the
breast cancer specimen are a general feature of tumors which
display elevated levels of glycolysis. The finding of significantly
lower CEdG in breast tumors relative to adjacent normal tissue can
potentially be explained by the observation that glycolytic cancers
possess lower levels of MG as a result of overexpression of the
glyoxalase system. This highly evolutionarily conserved system
consists of two non-homologous zinc metalloenzymes Glo1 and Glo2,
which act sequentially to convert MG into lactate using reduced
glutathione (GSH) as a catalytic cofactor.
[0077] Glo1/2 are overexpressed around 3-5.times. in many breast
cancers relative to normal mammary tissue, and enhanced expression
of either one or both enzymes has also been observed in prostate,
kidney, lung, colon, stomach, brain and ovarian cancers..sup.42,43
This is a metabolic adaptation to counter the pro-apoptotic effect
of MG accumulation in glycolytic tumors, which make Glo1 and Glo2
inhibitors attractive candidates for cancer therapeutics.
Accordingly, another application of the present quantitative
LC-MS/MS method is for monitoring the efficacy of glyoxalase
inhibitors, which would induce a dose dependent increase in CEdG
levels.
[0078] In sum, the new quantitative LC-MS/MS method for the
measurement of CEdG improves upon (with purity and volume) and
complements methods currently available for detecting protein AGEs,
and allows for a more comprehensive evaluation of the role of
nucleotide glycation in a wide range of human metabolic diseases,
including diseases in which CEdG levels affect the disease.
[0079] The foregoing merely illustrates various embodiments. As
such, the specific modifications discussed above are not to be
construed as limitations on the scope of the disclosed products and
methods. Equivalent embodiments are included within the
contemplated scope. All references cited herein are incorporated by
reference as if fully set forth herein.
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