1. 임신 중 흡연 노출 평가를 위한
바이오 마커 개발
진단검사의학과
곽호석

2. Tobacco smoke
• Tobacco smoke component: > 5,000 chemicals
• Electronic database of smoke
components: >2,000 chemicals
– Mainstream smoke: 542 chemicals
• Human inhalation risk: 98 chemicals
A list of smoke components needs to be selected with a sufficiently
broad chemical, toxicological, and pharmacological profile.

3. List of harzardous tobacco smoke components
Int. J. Environ. Res. Public Health 2011, 8

4. Int. J. Environ. Res. Public Health 2011, 8

5. Hazardous tobacco smoke component
• The list of Hoffmann analytes: biologically
or toxicologically active mainstream smoke
components
– Based on research from the early 1990s
– Only includes carcinogens

6. The list of Hoffmann analytes
Group Analytes Average Yields Group Analytes Average Yields
Ammonia (μg/cig) 11.02
1-Aminonaphthalene (ng/cig) 15.1 Hydrogen Cyanide (μg/cig) 109.2
Inorganics
Nitric Oxide (μg/cig) 223.41
Aromatic Amines 2-Aminonaphthalene (ng/cig) 10.3 Carbon Monoxide (mg/cig) 11.96
Organics Acrylonitrile (μg/cig) 8.28
3-Aminobiphenyl (ng/cig) 3
1,3-Butadiene (μg/cig) 29.94
4-Aminobiphenyl (ng/cig) 1.7 Benzene (μg/cig) 43.39
Volatile Hydrocarbons Isoprene (μg/cig) 297.68
Methyl ethyl ketone (μg/cig) 62.72
Toluene (μg/cig) 64.91
Acetaldehyde (μg/cig) 560.48 Styrene (μg/cig) 5.11
Acetone (μg/cig) 264.74 Pyridine (μg/cig) 7.02
Nitrogen Heterocyclics Quinoline (μg/cig) 0.23
Carbonyls Acrolein (μg/cig) 58.77
Nicotine (mg/cig) 0.75 (750ug)
Butyraldehyde (μg/cig) 29.58
Arsenic (ng/cig) 10.4
Crotonaldehyde (μg/cig) 16.18
Cadmium (ng/cig) 47.8
Formaldehyde (μg/cig) 21.61 Chromium (ng/cig) 73
Propionaldehyde (μg/cig) 43.92 Metals & Metalloids Lead (ng/cig) 33
Catechol (μg/cig) 37.9 Mercury (ng/cig) 3.82
Hydroquinone (μg/cig) 32.4 Nickel (ng/cig) 5.12
m+p-Cresol (μg/cig) 5.84 Selenium (ng/cig) 34.9
Phenols NAB (ng/cig) 16.3 (0.016ug)
o-Cresol (μg/cig) 1.89
Tobacco Specific NAT (ng/cig) 119 (0.119ug)
Phenol (μg/cig) 7.32
Nitrosamines (TSNAs) NNK (ng/cig) 115.6 (0.116ug)
Resorcinol (μg/cig) 0.91 NNN (ng/cig) 133.1 (0.133ug)
Polycyclic Aromatic Nicotine-free-dry-particulate-m
Benzo[a]pyrene (ng/cig) 7 'Tar' 8.91
Hydrocarbon (PAH) atter (mg/cig)

7. Biomarkers of tobacco smoke exposure
• Carbon monoxide (CO)
• Nicotine metabolite: Cotinine
• Polycyclic aromatic hydrocarbon (PAHs)
metabolite: 2-naphthol, 3-
hydroxyfluorenes, 1-hydroxypyrene
• Tobacco specific nitrosamines (TSNAs)
metabolite: NNAL

8. Tobacco-specific nitrosamines
(TSNAs)
 Processed tobaccos are invariably contaminated with TSNAs.
 Formed during processing (drying and fermentation)
 Not present in fresh and green tobaccos
 Different types of tobacco contain different concentrations of nitrosamines
 The higher the nitrate content of tobaccos the higher also the nitrosamine contamination
 The high-nitrate tobaccos or other dark tobacco types are high in TSNAs
 No correlation between the declared tar content of cigarettes and the TSNA
concentration in mainstream tobacco smoke
 No evidence that nicotine is nitrosated during the smoking process

9. Range for tar, nicotine and tobacco specific nitrosamine (TSNA)
in tobacco for commercial cigarettes from several countries

10. Results of testing for NNK yields from three
brands of cigarettes in various countries
Nicotine yield range
0.85 mg/cig to 1.3 mg/cig 0.68 mg/cig to 1.25 mg/cig
We see these results as a compelling and urgent argument for
government regulation of carcinogen concentrations in cigarettes.
Tobacco Control 2000;9:351–354

11. Chemical structure of TSNAs
Nitrosation
Nitrosamine structure

12. Formation of tobacco-specific
nitrosamines in tobacco

13. Potential third-hand smoke
PNAS ∣ April 13, 2010 ∣ vol. 107 ∣ no. 15

14. The metabolic activation and
inactivation of NNK
Steroid hormones
Drug: diuretics
Drug: block 11β-HSD
Drug
Drug: flavonoid
Flavanone
* 11β-HSD: 11β-hydroxysteroid dehydrogenase Similar structure: hormones

15. Chemical structure of NNAL
11β-HSD

16. Metabolism of NNK and NNAL and
formation of adducts

17. Structures of characterized
pyridyloxobutyl DNA adducts

18. Proposed activation pathways for
NNN, NNK and NNKOAc

19. Postulated metabolic pathway mediating
the toxicity of NNK
Other chemical
in smoke

21. Concentrations of total urinary 4-(methylnitrosamino)-
1-(3-pyridyl)-1-butanol (NNAL) (pg/mg creatinine) and cigarettes
per day in 1054 National Health and Nutrition Examination Survey
2007–2008 smokers

22. Adjusted geometric means in regression model with 95%
confidence intervals.

23. The metabolic activation and
inactivation of NNK
Steroid hormones
Drug: diuretics
Drug: block 11β-HSD
Drug
Drug: flavonoid
Flavanone
* 11β-HSD: 11β-hydroxysteroid dehydrogenase Similar structure: hormones

24. Relationships between Cigarette Consumption
and Biomarkers of Tobacco Toxin Exposure
CO 1-HOP (PAHs metabolite)
NNAL Cotinine

25. Structures of NNK, NNAL, and
NNAL-Gluc

26. Quantitation of Metabolites of NNK after
Cessation of Smokeless Tobacco Use
Smokeless tobacco Smoking
Time post-cessation (days)
NNAL t1/2: 10-18 days > cotinine t1/2 : 1days

27. Metabolizer phenotypes for smokers
• PM: poor metabolizer (ratio<2)
• IM: intermediate metabolizer
(ratio between 2 and 5)
• EM: extensive metabolizer (ratio >5)

28. Comparison of free, conjugated and total
urinary NNAL for 60 smokers

29. NNAL as a Biomarker of exposure to
cigarette smoke at the pregnancy
• Smoking patterns during pregnancy
• Second hand smoke during pregnancy
• Fetal exposure

30. Smoking patterns during pregnancy

31. Exposure to different sources of second-hand smoke during pregnancy
and its effect on urinary cotinine and NNAL concentrations

32. TOBACCO SMOKING OR EXPOSURE TO
SECOND-HAND SMOKE ON THE LEVELS OF
NNAL IN URINE OF MOTHER AND NEWBORN

33. Development of LC-MS/MS for
NNAL in urine
• Simple sample preparation
• Rapid analytical time
• High sensitivity

34. - Sample preparation
Enzyme treat Analysis LC-
• NNAL-glucuronides MS/MS
hydrolysis • Analytical time:
• Time: 20h 30min / test
Extraction Derivatization
• Liquid-Liquid • N-propyl
extraction derivatization
• Time: 1h • Time: 12h

35. LC-MS/MS analysis of NNAL
Blank Urine
LLOQ: 20 pg/mL

36. LC-MS/MS analysis of NNAL
Before enzyme
treatment
After enzyme
treatment
NNAL-glucuronides Concentration:
NNAL concentration (After enzyme treatment) – NNAL (Before enzyme treatment)

37. • Simple sample preparation
– Each detection of NNAL and NNAL-glucuronides
• Rapid analytical time
– Optimization of analytical column
• High sensitivity
– Optimization of sample preparation

38. The End