Tuesday, May 10, 2011
In order to prove the biological relevance of the above studies, the
authors took samples of fasting human urine, adjusted to a neutral pH,
and extracted the samples for CMdA following an extraction procedure
similar to that used for the D-glucose incubations. Mass spectrometry
confirmed the presence of CMdA with a peak of M+H+ 310 (Figure 1d). The peak was very strong, which was demonstrated by mixing experiments with synthesized CMdA sample.
Figure 4 - LC-MS spectrum using MRM spectrometric analysis of 1 M
HCL hydrolyzed: a) CMdA, b) calf thymus DNA, c) human serum DNA, and d)
CMdA and calf thymus DNA mixing experiment. The presence of CMA in DNA
samples was followed using a M+H+ 194 peak. Peaks from the
mixing experiment further confirmed the presence of CMA in both human
and calf thymus DNA samples. Experiments were repeated until findings
were proven conclusively.
DNA sample was hydrolyzed with 1 M HCL. This procedure cleaves off the purine bases from a DNA chain. CMdA itsef was hydrolyzed using 1 M HCL, and the sample was subjected to HPLC analysis (Figure 2b). The procedure completely hydrolyzed CMdA and CMA. An RT of 2.1 min was obtained. The identity of the peak for CMA M+H+ 194 was confirmed by electrospray MS-MS (Figure 2c and Figure 4a), including those from the synthesis reaction of CMA.
The presence of CMA in calf thymus DNA and human serum DNA samples was investigated next. Figure 4b depicts the presence of CMA in calf thymus DNA, while Figure 4c
shows its presence in human DNA (LC-MS mass spectrum using MRM mode
analysis), indicating that carboxymethylation of DNA nucleotide is
occurring in both of these DNA molecules. The presence of CMA in calf
thymus DNA was confirmed by carrying out mixing experiments with
hydrolyzed CMdA sample (Figure 4d). The experiments were
repeated, and modification of adenine nucleotide was observed both for
calf thymus and human serum DNA samples.
Glycoxidation and lipoperoxidation reactions produce permanent markers
on biomolecules such as proteins. These markers are an important
diagnostic tool to evaluate and quantify the extent of damage to the
host molecules and the physiological consequences resulting from the
presence of foreign materials in the molecules. Although important
biomarkers of protein modification of diabetes have been widely
established and used, continued investigations to detect and identify
biomarkers for diseases such as cancer are still being vigorously
pursued. DNA biomarkers in relation to diabetes and cancer will continue
to attract numerous technologies, methodologies, and procedures to
provide a better understanding of these diseases. 32Postlabeling20,21 mass spectrometry22–24 using electrochemical detection,25 fluorescence detection, and immunoassays are often used as tools in this field for quantification purposes.
Molecular damage can be largely eliminated by DNA repair processes. The
extent to which DNA adducts contribute to cancer development is an
important avenue of investigation. DNA adducts are an internal and
individual dosimeter of exposure of an organism to genotoxic compounds
and ultimately determine the biologically effective dose of a DNA
damaging chemical.26 According to Ref. 18, there is a direct
association between oxidative DNA damage and the complications of
diabetes, as demonstrated by measuring 8-oxo-2’-deoxyguanosine in the
urine and mononuclear cells of type II diabetic patients.18
Free radical modifications involving glycoxidation and lipoperoxidation reactions in proteins27,28
have direct consequences on disease processes in diabetes and
atherosclerosis, for example. Although there are striking similarities
in the functional natures of the amino acids present in the investigated
proteins, and biomolecules such as DNA, RNA, adenosine triphosphate
(ATP), and adenosine diphosphate (ADP), not much is known about the fate
of these substances in diabetic and other disease processes.
Experiments with 2’-deoxyadenosine and reactive carbohydrates such as
D-glucose, D-ribose, and L-ascorbic acid in the present investigations
indicate that glycoxidative reactions like those of proteins in diabetes
do indeed occur in the studied nucleoside. CMdA has been identified by
LC-MS (electrospray mass spectrometry experiments) from reactions of
2’-deoxyadenosine and D-glucose (Figure 1a) and 2’-deoxyadenosine and D-ribose (Figure 1b) incubations. CMdA showed a molecular ion (M+H+ 310) and fragment m/e
of 194, matching the breakdown of the parent ion into two groups at the
N–C bond between the heterocycle and the 2’-ribose part of the molecule
The identity of the CMdA compound produced from the reactions of
2’-deoxyadenosine with the carbohydrates D-glucose, D-ribose, and
L-ascorbic acid was achieved by synthesizing the CMdA compound using
2’-deoxyadenosine and iodoacetic acid raw materials. The HPLC and LC-MS
of the synthesized CMdA and the 2’-deoxyadenosine-carbohydrate incubated
CMdA samples (Figures 1a–c, e) were identical. The presence of identified carboxymethyl derivative (CMdA) in the urine samples (Figure 1d) of fasting subjects was also investigated, identified, and confirmed by the electrospray LC-MS experiment.
These experiments proved conclusively that CMdA is produced in human
systems. The results thus indicate that glycoxidative modifications
involving DNA modification (possibly also in RNA, unpublished results)
occur in our metabolic systems. CMdA may have arisen from glycoxidation
and lipoperoxidation reactions in DNA molecules that may have been
during repair of the DNA chain. Alternatively, CMdA possibly resulted
from glycoxidative modification of ADP, ATP, and AMP (adenosine
monophosphate) molecules present in a living system requiring energy
To confirm the presence of CMdA in the DNA chain itself, acid hydrolysis
of both calf thymus and human serum DNA samples was carried out. Prior
to that, CMdA was hydrolyzed with 1 M HCL, and the product gave a sharp peak at RT 2.1 min (Figure 1b) corresponding to CMA compared to the raw material CMdA in the HPLC peak at RT 7.1 min (Figure 1a). CMA gave a sharp peak at M+H+ 194 in LC-MS. The presence of CMA in the 1 M HCL hydrolyzed DNA (calf thymus and human samples) was investigated using LC-MS (Figure 4a–d).
All of these spectra and the mixing experiments of the DNA hydrolyzed
and identified CMA derivative confirmed that CMA was present in the DNA
samples investigated (Figure 4d).
Independent reports from investigations at a mass spectroscopy
laboratory reported a significant amount of modified CMdA in each
sample. The presence of CMdA compound in investigations of glycoxidation
or any other process in relation to human subjects has not been
reported thus far. These results prove conclusively that glycoxidation
and possibly lipoperoxidation reactions take place in human nucleic and
biomolecules such as those occurring in proteins. These findings are
important, since a modification will trigger removal of such a base from
the DNA chain and, if unsuccessful, may lead to mutations that will
affect many biological processes.
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- Lila, Z.A.; Mahtab, R. et al. Glycoxidative Modification of DNA-RNA in Relation to Diabetes. In The 1890 Land Grant System: AED/ARD Land Grant Conference; ARD Land Grant Conference, June 8–11, 2008, Memphis, TN, p 37.
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authors are with the Department of Biological and Physical Sciences,
South Carolina State University, 300 College St., Orangeburg, SC 29117,
U.S.A.; tel.: 803-516-4536; fax: 803-516-4685, e-mail: email@example.com,
with the exception of R. Idris, who is with the Department of Family
and Consumer Sciences, South Carolina State University. This work was
supported by the 1890 Evans-Allen Research Program, South Carolina State
University, and Project Export MUSC-SCSU (subcontract from NIH grant #5
P60 MD 000243). The authors wish to thank Dr. William E. Cotham of the
Mass Spectrometry Laboratory, USC (Columbia, SC) for performing the
LC-MS triple-quadrupole mass spectrometry analysis experiments.