1. Assessment of alcohol exposure
during early pregnancy

2. Drinking patterns among women
in baseline study

3. Estimation of alcohol exposure
Fetal FAEE
in meconium
?

4. Biologic markers of chronic alcohol abuse
(γGT)
(CDT)

5.  CDT
 Women tend to have higher than men.
 Pregnancy was found to be correlated with increased CDT
levels.
 γGT
 Nor is it a specific marker of chronic heavy alcohol use,
because other liver diseases or certain drugs also increase
serum levels of γGT.

6. Oxidative and non-oxidative pathways
of ethanol metabolism
Half life: 3 hr
in blood
PEthFAEE

7. Time courses for serum FAEE concentration and
ethanol concentration
JAMA (1996)
12 hr

8. Red blood cell fatty acid ethyl esters: a significant
component of fatty acid ethyl esters in the blood

9. Oxidative and non-oxidative pathways
of ethanol metabolism
Half life: 3 hr
in blood
Half life: 4 day
in blood
PEthFAEE

10. Daily alcohol intake for 21 days in group
Alcohol Clin Exp Res (1998)

11. Comparison of self reported ethanol consumption and true
positive results for different biomarkers

12. Box-plot of the alcohol markers grouped after reported
ethanol intake (g/day)
Alcohol & Alcoholism (2006)

13. Phosphatidylethanol and alcohol consumption in
reproductive age women

14. Estimation of alcohol exposure
Fetal FAEE
in meconium
?Mother PEth
in blood

15. First trimester of pregnancy
Measure of alcohol exposure
(PEth in blood)
FASD low
risk
FASD high
risk
Measure of
alcohol exposure
(FAEE in
meconium)
Purpose of Study
Birth
Early diagnosis of FASD
Early diagnosis
of FASD
PEth: HighPEth: Low

16. Analysis of
phosphatidylethanol(PEth)
by LC/MS/MS

17. Synthesis of Phosphatidylethanol
PEth

18. Structures of different PEth species

19. Sample Preparation
 Anders Helander, Molecular Species of the Alcohol Biomarker Phosphatidylethanol in
human Blood Measured by LC-MS, Clinical Chemistry, 55:7, 1395-1405 (2009)
1. 100 ul Blood + 600ul Isopropanol + 50ul Pprop (IS, 1.35 umol/L)
2. Mix for 10min.
3. Add Hexane (2 * 450 ul)
4. Mix for 10min.
5. Centrifuge for 10min (2000 g, 4℃)
6. Supernatants transfer to new glass tube
7. Evaporate under N2 gas at 30℃ using heating block
8. Dissolved in 50 ul hexane + 50 ul acetonitrile + 75 ul isopropanol
9. Transfer to 0.3 ml glass auto-sampler vial.

20. The End

21. LC-MS measurement of different PEth
molecular species in blood

22. Blood sample
 Blood collect into EDTA tubes
 Store at 4℃ for < 3days
 PEth is stable for at least 3 weeks in refrigerated
blood sample

23. LC-ESI-MS
 Negative ion mode
 MS condition
 Drying gas flow : 10.0 ψ
 Nebulizer gas : 20 ψ
 Drying gas temperature : 350 ℃
 Capillary voltage : 3000 V
 Column : C4 (50 mm Ⅹ 3 mm, 5um)

24.  LC system
 Gradient mode
 Sol A : 20% 2mmol/L ammonium acetate
80% acetonitrile
 Sol B : 100% isopropanol
LC-ESI-MS
Sol A
Sol B
2 4 6 8(min)
100(%)
50
0

25. LC-ESI-MS
• Flow rate : 200ul/min
• Injection volume : 10ul
• Reconditioning the column with
90% A and 10% B for 10min