Papers in Press. Published January 23, 2013 as doi:10.1373/clinchem.2012.193938
The latest version is at http://hwmaint.clinchem.org/cgi/doi/10.1373/clinchem.2012.193938
Clinical Chemistry 59:5
000 – 000 (2013)
Automation and Analytical Techniques
Quantification of 5-Methylcytosine and
5-Hydroxymethylcytosine in Genomic DNA from
Hepatocellular Carcinoma Tissues by Capillary
Hydrophilic-Interaction Liquid Chromatography/
Quadrupole Time-of-Flight Mass Spectrometry
Ming-Luan Chen,1 Fan Shen,2 Wei Huang,1 Jia-Hui Qi,2 Yinsheng Wang,3 Yu-Qi Feng,1*
Song-Mei Liu,2* Bi-Feng Yuan1*
BACKGROUND: 5-Methylcytosine (5-mC) is an important epigenetic modification involved in development
and is frequently altered in cancer. 5-mC can be enzymatically converted to 5-hydroxymethylcytosine (5hmC). 5-hmC modifications are known to be prevalent in DNA of embryonic stem cells and neurons,
but the distribution of 5-hmC in human liver tumor
and matched control tissues has not been rigorously
explored.
METHODS:
We developed an online trapping/capillary
hydrophilic-interaction liquid chromatography (cHILIC)/
in-source fragmentation/tandem mass spectrometry
system for quantifying 5-mC and 5-hmC in genomic
DNA from hepatocellular carcinoma (HCC) tumor
tissues and relevant tumor adjacent tissues. A polymerbased hydrophilic monolithic column was prepared
and used for the separation of 12 nucleosides by
cHILIC coupled with an online trapping system. Limits
of detection and quantification, recovery, and imprecision of the method were determined.
RESULTS:
Limits of detection for 5-mC and 5-hmC were
0.06 and 0.19 fmol, respectively. The imprecision and
recovery of the method were determined, with the relative SDs and relative errors being ⬍14.9% and 15.8%,
respectively. HCC tumor tissues had a 4- to 5-fold
lower 5-hmC content compared to tumor-adjacent tissues. In addition, 5-hmC content highly correlated
1
Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of
Education), Department of Chemistry, Wuhan University, Wuhan, China; 2 Center for Gene Diagnosis, Zhongnan Hospital of Wuhan University, Wuhan, China;
3
Department of Chemistry, University of California, Riverside, CA.
* Address correspondence to these authors at: Fax ⫹86-27-68755595; e-mail
bfyuan@whu.edu.cn, smliu@whu.edu.cn, yqfeng@whu.edu.cn.
M.-L. Chen and F. Shen contributed equally to this work.
Received July 30, 2012; accepted January 4, 2013.
Previously published online at DOI: 10.1373/clinchem.2012.193938
4
Nonstandard abbreviations: 5-mC, 5-methylcytosine; TET, ten– eleven translocation; 5-hmC, 5-hydroxymethylcytosine; 5-foC; 5-formylcytosine; 5-caC, 5-carboxylcytosine; LC-MS/MS, liquid chromatography/tandem mass spectrometry;
with tumor stage (tumor-nodes-metastasis, P ⫽
0.0002; Barcelona Clinic liver cancer, P ⫽ 0.0003).
CONCLUSIONS: The marked depletion of 5-hmC may
have profound effects on epigenetic regulation in HCC
and could be a potential biomarker for the early detection and prognosis of HCC.
© 2013 American Association for Clinical Chemistry
Methylation of DNA at the C5 position of cytosine to
give 5-methylcytosine (5-mC)4 is one of the bestcharacterized epigenetic modifications and has been
implicated in numerous biological processes, including embryogenesis, X-chromosome inactivation, genetic imprinting, and cellular differentiation (1, 2 ).
Properly established and maintained DNA methylation patterns are vital for mammalian development
and normal functions of the adult organism (3 ). Aberrant promoter methylation leading to inappropriate
transcriptional silencing or activation of genes is often
found in various types of human cancers (4 ). Although
DNA methylation has been viewed as a stable epigenetic mark, studies have revealed that this modification
is not as static as once thought. In fact, active DNA
demethylation has been observed and thoroughly studied in plants (5 ), but the mechanisms for active DNA
demethylation in mammalian cells remain elusive (6 ).
RPLC, reversed-phase liquid chromatography; ESI, electrospray ionization; CAD,
collision-activated dissociation; HILIC, hydrophilic-interaction liquid chromatograpoly(N-acryloyltris(hydroxymethyl)aminomethane-cophy;
NAHAM-co-PETA,
pentaerythritol triacrylate); qTOF-MS, quadrupole time-of-flight mass spectrometry;
HCC, hepatocellular carcinoma; dC, 2⬘-deoxycytidine; dG, 2⬘-deoxyguanosine; dA,
2⬘-deoxyadenosine; dI, 2⬘-deoxyinosine; 5-mdC, 5-methyl-2⬘-deoxycytidine;
5-hmdC, 5-hydroxymethyl-2⬘-deoxycytidine; ACN, acetonitrile; FA, formic acid;
TNM, tumor-nodes-metastasis; HBV, hepatitis B virus; FFPE, formalin-fixed, paraffinembedded; cHILIC, capillary HILIC; S/N, signal-to-noise; poly(MAA-co-EDMA), poly(methacrylic acid-co-ethylene glycol dimethacrylate); LOD, limit of detection; LOQ,
limit of quantification; RE, relative error; BCLC, Barcelona Clinic liver cancer; AUC,
area under the curve.
1
Copyright (C) 2013 by The American Association for Clinical Chemistry
The ten– eleven translocation (TET) proteins are capable of catalyzing the sequential oxidation of 5-mC to
5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5foC), and finally 5-carboxylcytosine (5-caC) (7–10 ). The
resulting 5-caC can be recognized and cleaved by
thymine-DNA glycosylase, thereby restoring unmethylated cytosine via base-excision repair machinery (11 ).
Thus, active DNA demethylation may be achieved
through a multistep oxidation of 5-mC with the generation of various forms of intermediates.
5-hmC has long been noted in bacteriophage
DNA, and its presence in mammalian cells was first
discovered in embryonic stem cells and adult neural
cells (7, 8 ). 5-hmC is also considered to play a crucial
role in cellular differentiation and pluripotency of embryonic stem cells (10 ). However, the biological significance of 5-hmC in human cancers remains elusive.
Mutations and decreased expression of TET genes display lower contents of 5-hmC in tumor tissues compared to healthy controls (12–14 ). In solid tumors,
5-hmC contents are reduced in the carcinomas of prostate, breast, liver, lung, pancreas, and colon, as revealed
by immunohistochemistry (12–14 ), as well as in lung
and brain tumors, as shown by liquid chromatography/
tandem mass spectrometry (LC-MS/MS) (15 ). These
findings suggest that decreased 5-hmC in genomic
DNA might be associated with tumor development.
5-hmC content in mammalian cells can be as low
as 0.009% of cytosine (molar ratio of 5-hmC/cytosine
in 293T cells) (9 ); therefore, a highly sensitive detection method is required for the quantitative analysis of
5-hmC content in mammalian genomes. Methods for
detecting 5-hmC in genomic DNA include radioactive
labeling followed by thin-layer chromatography detection (8 ), immunohistochemistry (16 ), HPLC (17 ),
LC-MS/MS (18 ), enzymatic glycosylation labeling
(19 ), and single-molecule real-time sequencing (20 ).
The thin-layer chromatography method involves labeling with radioactive isotope and the results are not
comparable to those of other available methods. Immunohistochemical staining is tedious and, to some
extent, less quantitative. HPLC analysis relies on chromatographic separation to avoid coelution with other
components. The glycosylation method is based on
enzymatic incorporation of modified glucose into
genomic 5-hmC; however, a complete enzymatic reaction may not be achieved, and 5-mC cannot be measured simultaneously. The measurement of 5-hmC by
single-molecule real-time sequencing is possible, but
the technology still needs improvements. Reversedphase liquid chromatography (RPLC) coupled with
MS/MS has been used for the analysis of 5-hmC
(9, 15, 18 ). However, an inherent weakness of RPLC is
that the high aqueous content of mobile phase results
in relatively low ionization efficiency during electros2
Clinical Chemistry 59:5 (2013)
pray ionization (ESI), which diminishes the detection
capability. Moreover, sodium adducts and in-source
collision-activated dissociation (CAD) fragmentation
often hamper the determination of target analytes
(21 ).
Hydrophilic-interaction liquid chromatography
(HILIC) has emerged as a technique complementary to
RPLC (22 ) owing to its good resolution for polar
compounds (23, 24 ). We previously fabricated a hydrophilic poly(NAHAM-co-PETA) monolith [poly(Nacryloyltris(hydroxymethyl)aminomethane-co-pentaerythritol triacrylate)] that had excellent column efficiency
and separation resolution toward nucleosides (25 ).
Furthermore, the employment of hydrophilic polymer– based monolith can enhance MS response of analytes owing to the high organic solvent– containing
mobile phase.
Here we report a system for the simultaneous detection of 5-mC and 5-hmC in genomic DNA by using
hydrophilic poly(NAHAM-co-PETA) monolith coupled with high-resolution quadrupole time-of-flight
mass spectrometry (qTOF-MS). Additionally, we used
an online trapping system that improved the detection
capability. We assessed 5-mC and 5-hmC contents in
143 hepatocellular carcinoma (HCC) tissues, which include 75 tumor tissues and 34 matched pairs of tumor
and adjacent tissues.
Materials and Methods
REAGENTS
We purchased 2⬘-deoxycytidine (dC), 2⬘-deoxyguanosine
(dG), 2⬘-deoxyadenosine (dA), thymidine (T), 2⬘deoxyinosine (dI), cytidine (C), guanosine (G), adenosine (A), uridine (U), inosine (I), and 5-methyl-2⬘deoxycytidine (5-mdC) from Sigma-Aldrich, and
5-hydroxymethyl-2⬘-deoxycytidine (5-hmdC) from
Berry & Associates. We prepared nucleosides in ACN/
H2O (99/1, vol/vol) at desirable concentration for the
construction of calibration curves and method validation.
We purchased HPLC-grade isopropanol and acetonitrile
(ACN) from Tedia and formic acid (FA) from Shanghai
Chemical Reagent. Water used throughout all experiments was purified by use of a Milli-Q water purification
apparatus (Millipore). All other reagents were obtained
from various commercial sources and were of analytical
grade unless otherwise indicated.
HEPATOCELLULAR CARCINOMA TISSUE SAMPLE COLLECTION
This study was approved by the ethics committee of
Zhongnan Hospital of Wuhan University. A total of
109 HCC patients [93 males and 16 females, mean (SD)
age 48.5 (12.0) years, range 18 – 80 years] were enrolled
from June 2005 to April 2011 at Zhongnan Hospital
of Wuhan University with TNM (tumor-nodes-
5-mC and 5-hmC in Hepatocellular Carcinoma
Fig. 1. Experimental setup for the analysis of nucleosides (5-hmdC, 5-mdC, dC, dG, dA, dI, T, C, G, A, U, and I) by
cHILIC-ESI-Q-TOF-MS.
metastasis) stage I (n ⫽ 73), stage II (n ⫽ 8), stage III
(n ⫽ 13), and stage IV (n ⫽ 15) cancer. Among them,
20 patients did not have hepatitis B virus (HBV) or
HCV infection and 89 patients had only HBV infection, including mild hepatitis (n ⫽ 23), moderate hepatitis (n ⫽ 19), severe hepatitis (n ⫽ 23), and liver
failure (n ⫽ 24); 71 patients did not have cirrhosis, 32
patients had compensated cirrhosis and 6 patients had
decompensated cirrhosis; 31 patients were drinkers. All
patient diagnoses were confirmed by pathology, and
patients underwent liver resection. We used a total of
143 formalin-fixed, paraffin-embedded (FFPE) tissue
samples, which included 34 pairs of tumor and
matched tumor-adjacent tissues as well as 75 tumor
tissues for which matched adjacent tissues were not
available (Supplemental Table 1, which accompanies
the online version of this article at http://www.
clinchem.org/content/vol59/issue5).
CAPILLARY HILIC-ESI-qTOF-MS SYSTEM
The capillary HILIC (cHILIC) was performed on a Shimadzu Prominence nano-flow liquid chromatography
system (Shimadzu) with two LC-20AD nano pumps, two
vacuum degassers, a LC-20AB HPLC pump, a SIL-20AC
HT autosampler, and a nano-flow control valve (Fig. 1).
We used an orthogonal-acceleration TOF mass
spectrometer (micrOTOF-Q; Bruker Daltonics) for
the cHILIC-MS experiment. The instrument was controlled by Bruker Daltonics Microcontrol software,
and Bruker Daltonics Data Analysis 3.4 software was
used for data analysis. Spectra were collected with a
time resolution of 1 s in the m/z range of 50 – 600. The
hydrophilic poly(MAA-co-EDMA) monolith [poly-
(methacrylic acid-co-ethylene glycol dimethacrylate)]
(1 cm, 50 ␮m inner diameter, 360 ␮m outer diameter)
was purchased from Weltech and used as online trapping columns. The poly(NAHAM-co-PETA) monolithic column (50 cm, 100 ␮m inner diameter, 360 ␮m
outer diameter) was prepared as previously described
(25 ) and used for the separation. The targeted compounds were separated on the poly(NAHAM-coPETA) monolithic column, which was connected to a
PicoTip™ (New Objective) nano-spray tip (360 ␮m
outer diameter, 10 ␮m inner diameter) with a zerodead-volume union (Upchurch Scientific) to minimize
postcolumn dead volume.
STATISTICAL ANALYSES
We performed all statistical analyses using SPSS 19.0
software (SPSS Inc.). All P values were two-sided, and P
values of ⬍0.05 were considered to be statistically significant. We estimated Pearson correlation coefficients for
each pair of covariate study and performed ROC analysis
to evaluate the ability of 5-mdC and 5-hmdC to discriminate tumor tissues from tumor-adjacent tissues.
Results
ESI-qTOF-MS DETECTION
The full-scan positive-ion ESI-MS of 5-mdC (online
Supplemental Fig. 1A) revealed the formation of [M ⫹
H]⫹, [M ⫹ Na]⫹, and [M ⫹ K]⫹ ions of the analyte at
m/z 242.1193, 264.0910, and 280.0710, respectively.
The in-source CAD fragment ion at m/z 126.0671 was
also observed. The corresponding ions were found for
5-hmdC at m/z 258.1093, 280.0912, 296.0651, and
Clinical Chemistry 59:5 (2013) 3
142.0614, respectively (see online Supplemental Fig.
1B). Previous reports indicated that protonated 5-mdC
tends to lose its ␤-D-2-deoxyribofuranose moiety to
give protonated 5-mC (21, 26, 27 ). Our results showed
that, under in-source CAD conditions, protonated
5-mdC and 5-hmdC can also lose the ␤-D-2deoxyribofuranose moiety to yield protonated 5-mC
and 5-hmC at m/z 126.0671 and 142.0614, respectively
(see online Supplemental Fig. 1, A and B). The abundance ratio for the protonated ion (I) of 5-mC (I126)
over that of 5-mdC (I242) was approximately 1/2.5, and
the corresponding ratio for 5-hmC at m/z 142.0614
(I142) over that of 5-hmdC at m/z 258.1093 (I258) was
approximately 5/4. These observations demonstrate
that the in-source CAD can result in a decrease in the
detection of 5-mdC and 5-hmdC. To circumvent this
problem, we optimized the in-source ESI-MS/MS conditions to stimulate the in-source CAD occurrence of
5-mdC and 5-hmdC. Under optimized in-source ESIMS/MS conditions, the I126 vs the I242 was approximately 10/1 for 5-mdC (see online Supplemental Fig.
1C), and the I142 vs the I258 was approximately 12/1 for
5-hmdC (see online Supplemental Fig. 1D). Neither
the [M ⫹ Na]⫹ nor the [M ⫹ K]⫹ ion was observed for
5-mC and 5-hmC. Therefore, with the optimized insource ESI-MS/MS conditions, we used the product
ions of 5-mdC (i.e., 5-mC, m/z 126.0671) and 5-hmdC
(i.e., 5-hmC, m/z 142.0614) for the identification and
quantification of cytosine methylation and hydroxymethylation, respectively. With this strategy, the detection capability for 5-mdC and 5-hmdC was ⬎1 order of
magnitude higher than before optimization. The detailed optimized conditions of ESI-MS/MS are shown
in the online Supplement.
ONLINE TRAPPING/cHILIC SYSTEM
We first optimized the separation conditions for the
above 12 nucleosides by changing the contents of ACN
(online Supplemental Fig. 2), FA (online Supplemental
Fig. 3), and isopropanol (online Supplemental Fig. 4)
in mobile phase. With the optimized mobile phase of
ACN/H2O/isopropanol/FA (90/5/5/0.02, vol/vol/vol/
vol), the 12 nucleosides could be baseline-resolved
within 30 min (online Supplemental Fig. 5).
Next, we investigated the influence of loading flow
rate (online Supplemental Fig. 6A), eluent volume (see
online Supplemental Fig. 6B), and washing volume
(see online Supplemental Fig. 6C) on the signal-tonoise (S/N) ratio of 5-mdC and 5-hmdC. Additionally,
we assessed the capacity of the online trapping
poly(MAA-co-EDMA) monolith in capturing 5-mdC
and 5-hmdC (see online Supplemental Fig. 6D). The
optimized conditions consisted of a loading flow rate of
10 ␮L/min, an eluent volume of 2250 nL, and a washing volume of 400 nL. In combination with the large
4
Clinical Chemistry 59:5 (2013)
injection volume (5 ␮L) to nanoscale separation system and the sample zone compression on the online
trapping column, the detection capability for 5-mdC
and 5-hmdC by cHILIC-ESI-qTOF-MS/MS was substantially improved without any apparent loss of separation resolution (Fig. 2A).
METHOD DEVELOPMENT
For the analysis of 5-mC and 5-hmC, extracted ion
chromatograms were obtained with 0.01-Da mass
width. We investigated the linearity of the method with
1.2 pmol dC standard supplemented with 5-mdC and
5-hmdC at different amounts ranging from 0.6 to 120
fmol (Table 1). With in-source ESI-MS/MS, we used
the MS peaks of the 5-mdC and 5-hmdC product ions,
5-mC and 5-hmC, for the identification and quantification of cytosine methylation and hydroxymethylation, respectively. We constructed the calibration
curves by plotting the mean peak area ratio of
5-mdC/dC or 5-hmdC/dC vs the mean molar ratio of
5-mdC/dC or 5-hmdC/dC on the basis of data obtained from triplicate measurements. The results
showed linearity within the range of 0.05%–10% (molar ratio of 5-mdC/dC or 5-hmdC/dC) with a coefficient value (R2) ⬎0.9979 (Table 1). Limits of detection
and quantification (LODs and LOQs) for 5-mdC and
5-hmdC were calculated as the amounts of the analytes
at S/N ratios of 3 and 10, respectively. The LODs and
LOQs were 0.06 and 0.20 fmol, respectively, for 5-mdC
and 0.19 and 0.64 fmol for 5-hmdC (Table 1). The
LODs for the 5-mdC and 5-hmdC obtained in this
study were, to the best of our knowledge, the lowest
compared to other previously reported methods with
mass spectrometry (9, 21 ).
We validated the method with the synthesized
5-mC- or 5-hmC-containing oligodeoxynucleotide by
comparing the measured 5-mdC or 5-hmdC content to
the theoretical 5-mdC or 5-hmdC content (online Supplemental Table 2). 5-mdC and 5-hmdC were determined from DNA hydrolysis product with CVs being
2.5%–11.0% and relative errors (REs) being ⫺16.4%–
13.0% (Tables 2 and 3), indicating that the cHILICESI-qTOF-MS method was reliable for the simultaneous determination of 5-mdC and 5-hmdC. We
evaluated the ion suppression by comparing the MS
intensities of 5-mdC and 5-hmdC in ACN/H2O and in
DNA hydrolysis products from synthesized DNA (online Supplemental Fig. 7). The peak areas of 5-mdC and
5-hmdC in ACN/H2O were 1.8% (0.2%) and 1.9%
(0.1%) greater than the peak areas in DNA hydrolysis
products from the synthesized DNA, suggesting that
ion suppression was negligible. The weak ion suppression for 5-mdC and 5-hmdC may be attributed to the
relatively clean DNA hydrolysis product as well as the
good chromatographic resolution of the analytes on ana-
5-mC and 5-hmC in Hepatocellular Carcinoma
Fig. 2. Extracted-ion chromatograms of nucleosides.
(A), Nucleoside standards obtained under the optimized conditions. (B), Nucleosides from 2 ng genomic DNA of HCC tissues.
Shown in the inset is the expanded chromatogram to reveal better the separation of 5-mdC, dC, C, dG, and 5-hmdC.
lytical monolithic column before mass spectrometry
analysis. In addition, we evaluated the imprecision and
recovery of the cHILIC-ESI-qTOF-MS/MS method (Tables 2 and 3). The relative SDs (CVs) and REs were
⬍14.9% and 15.8%, respectively.
MEASUREMENT OF 5-mC AND 5-hmC IN GENOMIC DNA FROM
HEPATOCELLULAR CARCINOMA TISSUES
With the developed cHILIC-ESI-qTOF-MS method,
we further investigated the minimal sample required
for the quantification of 5-mC and 5-hmC. We found
Table 1. Linearities, LOQs, and LODs for 5-mdC and 5-hmdC obtained by cHILIC-ESI-qTOF-MS/MS.
Regression line
Linear range
(vs [dC]), %
Slope
Intercept
R2
LOD, fmol
LOQ, fmol
5-mdC
0.05–10
0.0144 (0.0007)
0.0030 (0.0002)
0.9979
0.06
0.20
5-hmdC
0.05–10
0.0280 (0.0011)
0.0003 (0.0001)
0.9985
0.19
0.64
Analyte
Clinical Chemistry 59:5 (2013) 5
Table 2. Imprecision and recovery of the method for the detection of 5-mdC (n ⴝ 3 for each day).
Nominal [5-mdC]/[dC], %
0.05
0.10
0.20
0.40
0.80
1.00
2.00
Measured mean [5-mdC]/[dC], %
0.05
0.10
0.18
0.35
0.78
0.90
2.22
RSD, %a
9.8
3.6
4.7
5.3
3.3
4.6
5.2
⫺3.8
⫺4.8
⫺7.9
⫺12.2
⫺2.9
⫺8.1
10.6
4.00
Low QC High QC
(1.00)
(5.00)
6.00
8.00
10.00
3.87
5.57
7.62
11.01
0.94
4.7
6.3
5.4
9.2
3.4
4.9
⫺2.2
⫺6.5
⫺4.7
9.5
⫺6.0
⫺7.4
Day 1
RE, %
4.63
Day 1
Measured mean [5-mdC]/[dC], %
0.10
0.21
0.38
0.84
1.11
2.18
4.13
5.59
8.22
RSD, %
11.0
0.05
5.2
5.9
5.8
5.6
6.1
9.0
7.0
7.3
9.5
12.0
9.63
0.96
4.3
4.18
5.7
RE, %
⫺1.7
4.2
5.3
⫺4.9
4.3
10.5
9.0
3.3
⫺5.9
2.6
⫺4.2
⫺4.0
⫺16.4
2.22
Day 2
Measured mean [5-mdC]/[dC], %
0.05
0.11
0.20
0.43
0.84
0.90
4.38
5.87
RSD, %
8.2
6.6
8.9
8.2
6.3
7.3
10.6
7.7
8.3
10.6
8.41
12.1
9.23
RE, %
2.1
5.2
⫺2.7
6.4
5.1
⫺4.2
⫺1.8
11.2
⫺2.0
5.4
⫺7.6
0.06
1.07
5.09
6
7.7
7.0
1.8
5.01
Day 4
Measured mean [5-mdC]/[dC], %
0.11
0.21
0.45
0.79
0.91
2.12
3.48
6.61
11.07
1.09
RSD, %
10.1
7.2
8.1
6.9
4.9
5.2
7.5
8.7
9.9
13.1
7.33
14.9
4.9
4.1
RE, %
14.2
11.1
6.1
12.9
14.0
⫺11.0
5.3
⫺11.4
10.8
⫺8.9
10.5
9.0
0.2
1.13
4.95
Day 5
a
Measured mean [5-mdC]/[dC], %
0.06
0.11
0.43
0.89
1.19
2.02
RSD, %
9.9
6.4
11.8
0.22
7.6
5.1
5.7
9.2
11.6
10.8
12.1
13.2
7.3
6.2
RE, %
14.4
13.6
9.4
6.3
10.8
⫺0.9
⫺2.4
5.9
⫺7.3
⫺9.9
⫺9.4
13.0
⫺1.0
5.63
7.19
9.14
RSD, relative standard deviation.
that 5-mC could be quantified from 1 ng genomic
DNA, whereas 5-hmC could be quantified from 2 ng
genomic DNA. Figure 2B displays the extracted-ion
chromatogram of 12 nucleosides from the hydrolysis
product of 2 ng genomic DNA from HCC tissue (H009
tumor tissue) (see online Supplemental Table 1). The
detection of U and G was less sensitive than that of
other nucleosides, which may be attributed to the
weaker proton affinity of U and low elution efficiency
of G from the trapping column. The chromatograms of
5-mdC and 5-hmdC were extracted at m/z 126.0671
(0.01) and 142.0619 (0.01), respectively. The resolution of R5-mdC/dC and R5-hmdC/dG were ⬎1.5; therefore,
the presence of high contents of dC or dG does not
interfere with the quantification of 5-mdC and
5-hmdC.
5-hmC CORRELATES WITH TUMOR STAGES
A total of 143 HCC tissues derived from 109 patients,
including 75 tumor tissues and 34 pairs of matched
tumor and tumor-adjacent tissues, were analyzed by
cHILIC-ESI-qTOF-MS. The mean contents of 5-mC in
genomic DNA of all tumor tissues and all tumoradjacent tissues were 5.57% (0.83%) and 5.97%
(0.84%), respectively (Fig. 3A). The mean contents of
5-mC in genomic DNA from matched-pair tumor tissues and tumor-adjacent tissues were 6.00% (0.67%)
6
4.17
Clinical Chemistry 59:5 (2013)
and 5.97% (0.84%), respectively (Fig. 3C). The results
suggested there was no significant difference of 5-mC
between tumor tissues and tumor-adjacent tissues (Fig.
3, A and C). However, the 5-hmC content was markedly lower in genomic DNA of tumor tissues than
tumor-adjacent tissues. As shown in Fig. 3, B and D, the
mean contents of 5-hmC were 1.72% (0.45%) and
0.37% (0.13%) in tumor-adjacent tissues and tumor
tissues, respectively; the mean contents of 5-hmC in
genomic DNA from matched-pair tumor-adjacent tissues and tumor tissues were 1.72% (0.45%) and 0.42%
(0.19%), respectively.
We also compared 5-mC and 5-hmC contents
measured by cHILIC-ESI-qTOF-MS/MS and HPLCMS. The measured 5-mC and 5-hmC contents in tumor adjacent tissues were comparable with these two
methods, with REs being ⫺15.9% to 16.0% in all the
samples analyzed (online Supplemental Tables 3 and
4), indicating that the cHILIC-ESI-qTOF-MS method
is reliable for the determination of 5-mC and 5-hmC in
genomic DNA. However, the HPLC-MS cannot detect
5-hmC in tumor tissues (⬍1.0% vs [dC], as determined by cHILIC-ESI-qTOF-MS/MS) because of its
limited analytical sensitivity.
We performed statistical analysis to evaluate the
correlation of 5-mC and 5-hmC between tumor tissues
and tumor-adjacent tissues with respect to patients’
5-mC and 5-hmC in Hepatocellular Carcinoma
Table 3. Imprecision and recovery of the method for the detection of 5-hmdC (n ⴝ 3 for each day).
Nominal [5-hmdC]/[dC], %
0.05
0.10
Measured mean [5-hmdC]/[dC], %
0.05
0.10
RSD, %a
5.6
4.2
RE, %
4.0
2.0
0.20
Low QC High QC
(0.50)
(2.00)
0.40
0.80
1.00
2.00
4.00
6.00
8.00
10.00
0.23
0.40
0.81
1.06
2.25
4.09
6.64
7.91
10.92
0.44
9.9
7.6
6.8
5.9
3.5
2.6
3.6
4.9
5.9
3.3
4.0
15.8
0.8
0.8
5.6
12.6
2.5
9.7
⫺1.1
9.0
⫺11.4
⫺0.5
3.82
Day 1
1.99
Day 2
Measured mean [5-hmdC]/[dC], %
0.41
0.76
0.98
5.77
8.18
9.47
0.52
RSD, %
11.3
0.05
10.7
0.10
10.0
0.20
6.8
8.0
6.2
11.6
1.98
10.5
5.9
8.2
7.2
5.1
1.82
2.5
RE, %
⫺2.0
⫺3.0
⫺0.5
2.0
⫺4.7
⫺1.7
0.8
⫺5.0
⫺3.3
2.5
⫺5.0
3.2
⫺8.6
Day 3
Measured mean [5-hmdC]/[dC], %
0.05
0.11
0.20
0.40
4.11
6.24
8.09
11.06
0.47
RSD, %
6.6
4.6
7.5
8.6
10.2
0.81
1.04
7.2
10.5
2.00
8.3
4.2
7.6
5.2
6.4
2.18
7.8
RE, %
6.0
9.0
1.5
0.8
1.3
3.6
1.5
1.3
3.3
1.3
10.6
⫺6.6
9.4
Measured mean [5-hmdC]/[dC], %
0.05
0.09
0.18
0.38
0.81
1.08
2.12
3.80
5.57
7.81
9.79
0.53
RSD, %
5.6
6.5
6.2
7.2
5.2
6.3
3.2
7.6
3.2
4.6
6.2
7.0
8.4
⫺6.0
⫺7.0
⫺12.0
⫺4.6
1.4
8.4
5.0
⫺5.0
⫺6.7
⫺2.5
⫺2.0
6.2
5.2
Day 4
RE, %
2.10
Day 5
a
Measured mean [5-hmdC]/[dC], %
0.05
0.10
0.19
0.41
0.77
0.96
1.90
4.24
6.11
7.87
10.41
0.49
RSD, %
6.2
5.2
4.2
6.2
8.7
3.9
6.9
3.9
8.0
7.1
10.9
9.3
11.0
1.79
RE, %
4.0
⫺2.0
⫺5.5
1.4
⫺4.4
⫺3.8
⫺5.4
5.3
2.0
⫺1.3
4.0
⫺1.8
⫺10.4
RSD, relative standard deviation.
age, sex, tumor stages [TNM and Barcelona Clinic liver
cancer (BCLC)], liver inflammation stages, liver cirrhosis stages, and liver function parameters (alanine
transaminase, aspartate aminotransferase, total protein, albumin, globulin, ␥-glutamyl transpeptidase,
and alkaline phosphatase). On the basis of Pearson correlation coefficient, 5-hmC content was significantly
correlated with tumor stages (TNM, r ⫽ ⫺0.324, P ⫽
0.0002; BCLC, r ⫽ ⫺0.338, P ⫽ 0.0003) (online Supplemental Table 5). As shown in Fig. 3, E and F, decreased 5-hmC was associated with tumor progression.
However, there was no correlation of 5-hmC with respect to age (P ⫽ 0.149), sex (P ⫽ 0.190), liver inflammation stages (P ⫽ 0.915), liver cirrhosis stages (P ⫽
0.117), alcohol use (P ⫽ 0.068) (see online Supplemental Table 5), or liver function parameters (data not
shown). Additionally, 5-mC was not associated with
tumor stages (TNM, P ⫽ 0.765; BCLC, P ⫽ 0.681), age
(P ⫽ 0.979), sex (P ⫽ 0.586), liver inflammation stages
(P ⫽ 0.739), liver cirrhosis stages (P ⫽ 0.567), alcohol
use (P ⫽ 0.056) (see online Supplemental Table 5), or
liver function parameters (data not shown).
We further evaluated the possibility of 5-hmC as a
biomarker for the early detection and prognosis of human HCC by performing ROC analysis. As shown in
Fig. 3G, 5-hmC was highly effective in the detection of
HCC, with the area under the curve (AUC) being
0.969; however, 5-mC was not appropriate for the detection of HCC, with AUC being 0.599 (Fig. 3H).
Discussion
HCC is one of the most common human cancers (28 ).
Asians have a high risk of HCC development (29 ). The
importance of epigenetic alterations in human HCC,
however, has not been rigorously explored despite a
few reports suggesting changes in global cytosine methylation and hydroxymethylation in cancer cells
(12, 14, 16, 30, 31 ).
DNA methylation plays an important role in tumor pathogenesis, and promoter CpG island hypermethylation in tumor-suppressor genes is a common
hallmark of human cancers (1, 4, 32 ). However, it is
still unclear why certain regions become hypermethylated and others remain unmethylated. Hypomethylation at promoters can activate the aberrant expression
of oncogenes (33 ). The discovery that 5-mC can be
oxidized to 5-hmC by TET enzymes has raised many
questions regarding the role of 5-hmC in epigenetic
reprogramming. A role for 5-hmC as an intermediate
in DNA demethylation has been postulated (7–11 ). A
recent report has shown that the rapid loss of 5-mC
from mouse paternal pronuclei was accompanied by
an accumulation of genome-wide 5-hmC (34 ). HowClinical Chemistry 59:5 (2013) 7
Fig. 3. Quantification and statistical analysis of 5-mC and 5-hmC in human HCC tumor tissues and tumor-adjacent
tissues.
(A), 5-mC content in HCC tumor tissues and tumor-adjacent tissues. (B), 5-hmC content in HCC tumor tissues and tumoradjacent tissues. (C), 5-mC content in matched-pair HCC tumor tissues and tumor-adjacent tissues. (D), 5-hmC content in
matched-pair HCC tumor tissues and tumor-adjacent tissues. (E), Correlation of 5-hmC content with human HCC tumor TNM
stages. (F), Correlation of 5-hmC content with human HCC tumor BCLC stages. (G), ROC curve for 5-hmC score for human HCC
tumor tissues. (F), ROC curve for 5-mC score for human HCC tumor tissues. AUC [mean (SD)] are shown.
ever, the failure to find many of the predicted intermediates of an active oxidative demethylation pathway of
normal mouse tissues challenges the existence of such a
mechanism (9, 35 ), which may be attributed to the lack
of highly sensitive methods for the detection of
intermediates.
We developed a method for simultaneous determination of 5-mC and 5-hmC by cHILIC-ESI-qTOFMS/MS. The highly sensitive method allowed for the
determination of low contents of 5-mC and 5-hmC
with a DNA sample of only 2 ng. With this method, we
provided evidence of lower content of 5-hmC in HCC
by analyzing matched-pair tumor tissues and tumoradjacent tissues. Because 5-mC is required as a substrate for oxidation to generate 5-hmC, the decrease in
5-hmC could emanate from reduced 5-mC in tumor
tissues. To examine this possibility, we also analyzed
the 5-mC contents in genomic DNA from tumor tissues and tumor-adjacent tissues. The genome-wide
5-mC content was similar between tumor tissues and
tumor-adjacent tissues (Fig. 3), revealing that the diminished contents of 5-hmC in HCC tissues is not due
to decreased contents of global cytosine methylation.
8
Clinical Chemistry 59:5 (2013)
Our correlation analysis also showed that 5-hmC
correlated with tumor stage (Fig. 3, E and F), whereas
no such association was found for 5-mC. In addition,
ROC analysis suggested that HCC can be characterized
by the change of 5-hmC but not 5-mC (Fig. 3, G and
H). The discovery that 5-hmC contents were reduced
in HCC, together with previous reports of the decreased contents of 5-hmC in other types of cancer tissues (15, 16 ), suggests that the depletion of 5-hmC
could be a general feature of solid tumors. The biological significance of the loss of 5-hmC in tumors remains
to be elucidated; nevertheless, loss of 5-hmC could be
used as biomarker for the early detection and prognosis
of HCC.
Author Contributions: All authors confirmed they have contributed to
the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design,
acquisition of data, or analysis and interpretation of data; (b) drafting
or revising the article for intellectual content; and (c) final approval of
the published article.
5-mC and 5-hmC in Hepatocellular Carcinoma
Authors’ Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form.
Disclosures and/or potential conflicts of interest:
Employment or Leadership: None declared.
Consultant or Advisory Role: None declared.
Stock Ownership: None declared.
Honoraria: None declared.
Research Funding: Y. Wang, NIH (R01 DK082779); Y.-Q. Feng, the
National Basic Research Program of China (973 Program)
(2012CB720601), the National Natural Science Foundation of China
(91017013, 31070327); S.-M. Liu, the National Basic Research Program of China (973 Program) (2012CB720601), the National Natural Science Foundation of China (81271919); B.-F. Yuan, the Fundamental Research Funds for the Central Universities, the National
Basic Research Program of China (973 Program), the National Natural Science Foundation of China (21205091, 21228501), the Natural
Science Foundation of Hubei Province (2011CDB440).
Expert Testimony: None declared.
Role of Sponsor: The funding organizations played no role in the
design of study, choice of enrolled patients, review and interpretation
of data, or preparation or approval of manuscript.
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