Tuesday, May 10, 2011
Identification of CMdA in urine sample
For analysis, a fasting urine sample (1 mL) was adjusted to pH 7.0 and
dried (<60 °C) in a rotary evaporator. Methanol (3 mL) was added to
the dried residue; the mixture was sonicated, vortexed, and centrifuged;
and the methanol fraction was collected. The process of extraction with
3 mL methanol was repeated, and the combined methanol fractions were
subjected to HPLC and LC-MS. Figure 1d shows a molecular ion (M+H+ 310) and fragment m/e of 194.
Figure 2 - HPLC analysis of a) CMdA, b) 1 M
HCL hydrolyzed CMdA, and c) TIC and mass spectrum (electrospray MS-MS
product ion scan) of CMA. Conversion of CMA and CMdA (HPLC [a]: RT 7.1
min) to CMA (HPLC [b]: RT 2.1 min) involves complete conversion of the
CMdA to the CMA compound. LC-MS (electrospray MS-MS product ion scan) of
the compound gave M+H+ 194 and fragments at m/e 135, 107 attributed to the CMA compound.
3. Hydrolysis of CMdA: 1 M HCl was added to CMdA at 27 °C for 24
hr. The sample was evaporated at 60 °C until dry using a rotary
evaporator. Methanol (3 mL) was added to the dried residue; the mixture
was sonicated, vortexed, and centrifuged; and the methanol fraction was
collected. The sample was then analyzed by HPLC (Figure 2b) (RT: 2.1 min) and electrospray LC-MS (MS-MS ion scan). Figure 2c shows a molecular ion (M+H+ 194) and fragment m/e of 135.
Synthesis of carboxymethyladenine (CMA) using adenine and iodoacetic
acid: A mixture of adenine (13.5 mg, 0.1 mmol) and iodoacetic acid
(threefold) (54 mg, 0.1 mmol) in 0.2 M Pi (1 mL), pH 7.4, 37 °C
was incubated for 7 days; the pH was adjusted to 7.0; the sample was
dried; and the methanol extract was subjected to HPLC and LC-MS
(electrospray, MS–MS ion scan). A molecular ion (M+H+ 194) and fragment m/e of 135 were found.
Hydrolysis of calf thymus DNA (CTDNA) and human serum DNA: CTDNA (10
μg/μL) and human serum DNA (10 μg/μL) samples were hydrolyzed with 1 M
HCl at 27 °C for 24 hr in separate experiments. Methanol (3 mL) was
added to the dried residues; the mixtures were sonicated, vortexed, and
centrifuged; and the methanol fractions were collected. The methanol
extracted samples were then analyzed by HPLC and LC-MS using multiple
reaction monitoring (MRM) mode (electrospray) for analysis, giving an
ion (M+H+ 194) and fragment m/e of 135 for both experiments.
Analysis by HPLC
HPLC analysis was performed as described above, along with a
water/acetonitrile solvent mixture using gradient analysis. Solvent A
was 100% water; solvent B was 100% acetonitrile. The flow rate was 1
Mass spectrometry analysis
LC-MS triple quadrupole mass spectrometry (electrospray, MS-MS MRM mode)
was used for detection of the CMdA and CMA from incubated and DNA
3 - Reaction scheme summarizing various interconversion experiments,
synthetic protocols leading to the detection of CMdA in fasting human
urine, and CMA in calf thymus and human serum DNA samples.
The results of the experiments described in this investigation are shown in the reaction scheme in Figure 3.
These include reactions of the nucleotide dA with carbohydrates such as
D-glucose, D-ribose, and L-ascorbic acid producing CMdA; its detection
in urine experiments; identification of CMA in human and calf thymus
DNA; and related experiments.
Since DNA molecules are made up of units of nucleotides, it is expected
that any research on glycoxidation reaction with these individual units
will enable researchers to find out whether DNA polymer is involved at
all in such reactions. With this concept in mind, the authors began
their studies with the purine nucleotide 2’-deoxyadenosine as starting
point. This was incubated with D-glucose, D-ribose, and L-ascorbic acid
at pH 7.4 and 37 °C using 0.2 M phosphate buffer. Since dA is
soluble in methanol, extraction of the dried incubated samples led to
the isolation of the modified nucleotide from experiments carried out in
the phosphate buffer system. The samples from methanol extractions were
then examined by HPLC analysis using gradient elution.
LC-MS is widely used for the detection and quantitation of nucleotide
samples from DNA. It was also used to identify the presence of CMdA
(Figures 1a–c) and the synthesis reaction of CMdA (Figure 1e). The ion plot monitoring M+H+ 310 ions was clearly visible in the synthetic samples as well as D-glucose (Figure 1a), D-ribose (Figure 1b), L-ascorbic acid (Figure 1c), and synthetic samples (Figure 1e). The electrospray MS spectrum of the synthetic CMdA (Figure 1e) shows the fragmentation at the N–C bond of the heterocycle and the deoxyribose moiety in the CMdA compound.