SOFT 2004 Meeting Abstracts - Society of Forensic Toxicologists

A77
DETERMINING THE USE OF N1-ETHYL· GUANINE AS A BIOMARKER· FOR FATAL
ALCOHOL SYNDROME
Stacy L. Gelhaus', William R. LaCourse, Janine Cook, Katherine Squibb
Department ofChemistry and Biochemistry, University of Maryland, Baltimore County, Baltimore,
Maryland, USA
Background: Fetal alcohol syndrome (FAS) and fetal alcohol spectrum disorders (FASD) represent the
largest categories of preventable mental retardation syndromes and birth defects. It is theorized that
ethanol's toxicity is in part mediated by damage to DNA. In DNA, ethanol's primary metabolite,
acetaldehyde forms an N2-ethylguanine DNA adduct. The duration of this adduct, its affect on DNA
replication, and its repair mechanism are currently unknown. A case study is presented to assess N 2 _
ethylguanine as a potential alcohol biomarker with possible clinical utility in predicting the risk of
FASIFASD in infants born to alcohol-using women.
Methods: The case subject was a 20 year old who reported binge drinking on hard alcohol for the first 5
weeks of her pregnancy. The separation and quantification of N 2 -ethylguanine from unadducted
nucleosides was performed by an in-house protocol. DNA was purified from whole blood using a
commercial genomic DNA purification kit. The isolated DNA was completely hydrolyzed into monomer
bases using established digestion procedures to liberate purine bases; recovery of the purine bases was
>96%. The N 2 -ethylguanine DNA adduct was isolated and quantified by reversed phase HPLC with
isocratic elution and UV/fluorescence detection. Bases were identified by retention time and by standard
spiking. Base quantitation was by calibration curves ofstandard peak height signals.
Results: The identification of N 2 -ethylguanine by HPLC was confirmed through standard spiking and
LCIMS. HPLC precision for the N2-ethylguanine was 0.2% for peak area or 3.9% for peak height.
Standard curve linearity was to 15 ~mol (r = 0.9999). Preliminary analytical sensitivity was 29 nmol
(HPLC/uV detection), 2.9 nmol (HPLClfluorescence detection), and 20 fmol (LC/MS). No interference
was found from free bases. The subject's N 2 -ethylguanine concentration determined by LCIMS at 20 weeks
gestation was 11.7 pmol/mg DNA, which declined to 8.3 pmol/mg DNA by 30 weeks. Her adduct levels
are higher than a comparison nonpregnant social drinker whose N 2 -ethylguanine concentration was 6.9
pmol/mg. Reproducibility of these initial subject results is pending. The subject's fullterm daughter was in
the 75th percentile for weight, 17th percentile for length and 10th percentile for head circumference, an
indicator ofFAS.
Conclusions: Initial studies indicate the acceptability of this method for the separation and detection of
purines, including the N 2 -ethylguanine. Additional subject data is needed to confirm the adduct's clinical
utility.
Public Health implications: Clinical validation ofN2-ethylguanine as a biomarker for alcohol damage will
fortify the established panel of alcohol biomarkers, aiding in the identification of pregnancies at risk for
FASIFASD and elucidation ofthe mechanisms of alcohol pathology.
Keywords: DNA, Biomarker, FAS
Page 191