Assessment of alcohol exposure
during early pregnancy
Drinking patterns among women
in baseline study
Estimation of alcohol exposure
Fetal FAEE
in meconium
?
Biologic markers of chronic alcohol abuse
(γGT)
(CDT)
 CDT
 Women tend to have higher than men.
 Pregnancy was found to be correlated with increased CDT
levels.
 γGT
 Nor ...
Oxidative and non-oxidative pathways
of ethanol metabolism
Half life: 3 hr
in blood
PEthFAEE
Time courses for serum FAEE concentration and
ethanol concentration
JAMA (1996)
12 hr
Red blood cell fatty acid ethyl esters: a significant
component of fatty acid ethyl esters in the blood
Oxidative and non-oxidative pathways
of ethanol metabolism
Half life: 3 hr
in blood
Half life: 4 day
in blood
PEthFAEE
Daily alcohol intake for 21 days in group
Alcohol Clin Exp Res (1998)
Comparison of self reported ethanol consumption and true
positive results for different biomarkers
Box-plot of the alcohol markers grouped after reported
ethanol intake (g/day)
Alcohol & Alcoholism (2006)
Phosphatidylethanol and alcohol consumption in
reproductive age women
Estimation of alcohol exposure
Fetal FAEE
in meconium
?Mother PEth
in blood
First trimester of pregnancy
Measure of alcohol exposure
(PEth in blood)
FASD low
risk
FASD high
risk
Measure of
alcohol e...
Analysis of
phosphatidylethanol(PEth)
by LC/MS/MS
Synthesis of Phosphatidylethanol
PEth
Structures of different PEth species
Sample Preparation
 Anders Helander, Molecular Species of the Alcohol Biomarker Phosphatidylethanol in
human Blood Measur...
The End
LC-MS measurement of different PEth
molecular species in blood
Blood sample
 Blood collect into EDTA tubes
 Store at 4℃ for < 3days
 PEth is stable for at least 3 weeks in refrigerat...
LC-ESI-MS
 Negative ion mode
 MS condition
 Drying gas flow : 10.0 ψ
 Nebulizer gas : 20 ψ
 Drying gas temperature : ...
 LC system
 Gradient mode
 Sol A : 20% 2mmol/L ammonium acetate
80% acetonitrile
 Sol B : 100% isopropanol
LC-ESI-MS
S...
LC-ESI-MS
• Flow rate : 200ul/min
• Injection volume : 10ul
• Reconditioning the column with
90% A and 10% B for 10min
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임신초기알코올노출평가(곽호석)

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임신초기알코올노출평가(곽호석)

  1. 1. Assessment of alcohol exposure during early pregnancy
  2. 2. Drinking patterns among women in baseline study
  3. 3. Estimation of alcohol exposure Fetal FAEE in meconium ?
  4. 4. Biologic markers of chronic alcohol abuse (γGT) (CDT)
  5. 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. 6. Oxidative and non-oxidative pathways of ethanol metabolism Half life: 3 hr in blood PEthFAEE
  7. 7. Time courses for serum FAEE concentration and ethanol concentration JAMA (1996) 12 hr
  8. 8. Red blood cell fatty acid ethyl esters: a significant component of fatty acid ethyl esters in the blood
  9. 9. Oxidative and non-oxidative pathways of ethanol metabolism Half life: 3 hr in blood Half life: 4 day in blood PEthFAEE
  10. 10. Daily alcohol intake for 21 days in group Alcohol Clin Exp Res (1998)
  11. 11. Comparison of self reported ethanol consumption and true positive results for different biomarkers
  12. 12. Box-plot of the alcohol markers grouped after reported ethanol intake (g/day) Alcohol & Alcoholism (2006)
  13. 13. Phosphatidylethanol and alcohol consumption in reproductive age women
  14. 14. Estimation of alcohol exposure Fetal FAEE in meconium ?Mother PEth in blood
  15. 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. 16. Analysis of phosphatidylethanol(PEth) by LC/MS/MS
  17. 17. Synthesis of Phosphatidylethanol PEth
  18. 18. Structures of different PEth species
  19. 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. 20. The End
  21. 21. LC-MS measurement of different PEth molecular species in blood
  22. 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. 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. 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. 25. LC-ESI-MS • Flow rate : 200ul/min • Injection volume : 10ul • Reconditioning the column with 90% A and 10% B for 10min

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