TITLE: Teeth Steroid Extraction for &#34;Steroid profiling in human primary teeth via liquid chromatography-tandem mass spectrometry for long-term retrospective steroid measurement&#34;
AUTHORS: Ruolan S. Wu, hamden@zoology.ubc.ca, Melody Salehzadeh, Michael X. Li, Asmita Poudel, Kim L. Schmidt, Michael S. Kobor, Kiran K. Soma

[DESCRIPTION]
Steroid hormones are important modulators of many physiological processes, and
measurements of steroids in blood, saliva, and urine matrices are widely used to
assess endocrine pathologies and stress. However, these matrices cannot be used to
retrospectively assess early-life stress and developmental endocrine pathologies,
because they do not integrate steroid levels over the long term. A novel biological
matrix in which to measure steroids is primary teeth (or “baby teeth”). Primary teeth
develop early in life and accumulate various endogenous molecules during their
gradual formation. Here, we developed and validated the first assay to measure
steroids in human primary teeth using liquid chromatography-tandem spectrometry
(LC-MS/MS). Our assay is highly sensitive, specific, accurate, and precise. It allows for
the simultaneous quantification of 17 steroids in primary teeth (16 of which have not
been examined previously in primary teeth). Overall, steroid levels in primary teeth
were relatively low, and 8 steroids were quantifiable. Levels of
dehydroepiandrosterone, cortisol, and progesterone were the highest of the 17 steroids
examined. Next, we used this assay to perform steroid profiling in primary teeth from
males and females. The same 8 steroids were quantifiable, and no sex differences
were found. Levels of androgens (androstenedione and testosterone) were positively
correlated, and levels of glucocorticoids (cortisol, cortisone, corticosterone, 11-
dehydrocorticosterone) were also positively correlated. These data demonstrate that
multiple steroids can be quantified by LC-MS/MS in human primary teeth, and this
method potentially provides a powerful new way to retrospectively assess early-life
stress and developmental endocrine pathologies.

[STEPS]
SECTION: First Day
1.  
Put 5 1.4mm  beads in a 2mL Bead ruptor tube
 
SECTION: First Day
2. Label bead ruptor tubes
SECTION: 2nd Day
15. Preheat speed vac to 60C
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3. Label 0.6mL polypropelene microcentrifuge tubes to
correspond with bead ruptor tubes
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4. Label LC-MS/MS vials to corresond with bead ruptor tubes and
put glass inserts in vials
SECTION: First Day
5. Make 50% MeOH
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6. Make 25% MeOH 
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7. Use previously
diluted standard curve. 
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8. Use previoulsy made 5X IS.
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9. Make enough 1X I.S. to use for this asay 
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10. Preheat oven to 60C
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11. Rinse all 12x75mm Fisher culture tubes needed for experiment
with 1mL MeOH, vortex for 2sec, dump MeOH waste into waste beaker, add another
1mL MeOH to each tube, vortex for 2 sec, dump MeOH into waste and place
inverted tubes in tube rack in drying oven at 60C 
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12. Label rinsed and dried culture tubes
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13. Cover all rinsed, dried and labelled tubes (right-side up)
with aluminum foil ensuring complete covering of tubes
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14. Wash all teeth to remove saliva and blood
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16. Remove all reagents from 4C or -20C and allow to come to
room temp
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17. Retrieve the cleaned pieces of three teeth from the drawer
and record the weight
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18. Crush each piece into powder with the tooth crusher
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19. Collect and weigh the powder
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20. Put all tooth powder into corresponding bead ruptor tube
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21. Add 10ul STD to appropriate bead ruptor tubes
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22. Add 1mL room temp HPLC ACN to all tubes
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23. Add 50ul I.S. to all tubes excpet BLKBLK
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24. Add 50uL of 50% MeOH to BLKBLK
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25. Vortex 2sec
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26. Homogenize 4m/s for 30sec
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27. Centrifuge at 16,100g for 5min
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28. Remove 1000ul supernatant (94%) and put in 12x75mm culture
tube
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29. Add 500ul Hexane to all samples
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30. Vortex 5sec
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31. Centrifuge at 3200g for 2min
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32. Remove hexane and put in waste
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33. Dry ACN at 60C for 45min
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34. Resuspend in 55ul 25% MeOH
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35. Vortex 5 sec
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36. Centrifuge 3200g for 1min 
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37. Transfer all supernatant to 0.6mL centrifuge tube
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38. Centrifuge 16100g for 2min
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39. Transfer 50ul supernatant to LC insert using gel loading
tips
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40. Store in -20C until injection