Converting to 11-hydroxy

[cyclopath]

first you prep your livers…

PREPARATION OF MICROSOMES FROM HUMAN LIVER

Microsomes can be prepared from human or laboratory animal liver tissue that is either fresh or snap frozen in liquid nitrogen, and both tissues and microsomes can be stored up to 5 years at −70°C (Yamazaki et al., 1997). In our experience, CYP and UGT activities in human liver microsomes (HLM) are stable for at least 10 years when stored at −70°C (unpub. observ.). This protocol for the preparation of HLM is a slightly modified version of one first reported by Bowalgaha et al. (2005). Additionally, HLM can be

SUPPORT PROTOCOL 1

Pharmacokinetics

7.8.11

Current Protocols in Pharmacology

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Drug Metabolism Using Liver Microsomes

7.8.12

characterized for the measurement of P450 content and microsomal yield (see Support Protocol 2). Microsomes from a variety of human tissues and laboratory animal tissues and species can be purchased from commercial suppliers such as Corning Life Sciences (http://www.corning.com), Bioreclamation/IVT (http://www.bioreclamationivt.com), or XenoTech (http://www.xenotech.com).

Materials

Human liver tissue (1 gm of tissue per 5 ml of microsome preparation buffer) Microsome preparation buffer (see recipe)
Microsome storage buffer (see recipe)

Open-top polycarbonate centrifuge tubes (50 ml)
Surgical scissors
Tissue homogenizer (e.g., Ultra Turrax T25, http://www.ika.com) Potter-Elvehjem tissue grinder (30 ml) with Teflon pestle (motor driven) Refrigerated centrifuge
Syringe (50 ml) with blunt-end aspiration needle
Ultracentrifuge tubes (􏰂25 ml; e.g., Beckman-Coulter)
Ultracentrifuge, refrigerated to 4°C before use
Ultracentrifuge rotors, maintained at 4°C for at least 16 hr before use. Plastic round-ended probe
Potter-Elvehjem tissue grinder (10 ml) with Teflon pestle (hand held) Glass storage vials (suitable for −70°C)

Additional reagents and equipment for determining protein concentration (APPENDIX 3A; Olson, 2016)

Prepare tissue

When preparing HLM it is essential to precool the equipment and to maintain, using ice buckets, the liver tissue, solutions, centrifuge tubes, and grinders/homogenizers at 4°C. A full-face shield and protective clothing, including gloves, must be worn when working with human tissue and high-speed homogenizing equipment.

Weigh frozen liver tissue into clean (labeled) open-top 50-ml polycarbonate cen- trifuge tube(s) containing 5 ml of ice-cold microsome preparation buffer per g of liver tissue. Allow tissue to thaw on ice.

Finely mince the liver tissue with surgical scissors.

Insert the tip of the mechanical tissue homogenizer (e.g., Ultra Turrax T25 set at 20,500 rpm) into the tissue solution twice, each time for 30 sec, with a 30-sec cooling period between bursts.

If using less than 1 g of human liver tissue, increase the volume of the microsome preparation buffer to ensure that the tip of the tissue homogenizer probe is covered. This will enable homogenization without frothing.

The time and number of bursts required for mechanical disruption of tissue will vary (e.g., 2 to 4 30-sec bursts) for certain tissues such as human kidney and dog and monkey liver, which are more fibrous than rat and mouse liver.

Transfer the ground liver solution into an ice-cold 30-ml Potter-Elvehjem grinder. Homogenize using eight full strokes using the matched motorized Teflon pestle.

A minimum of two full strokes is necessary to ensure sufficient homogenization. Increasing the number of strokes increases the yield of microsomes, but prolonged shear force and possible heating during this step can cause contamination of the homogenate by other proteins and possible degradation of microsomal enzymes.

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Current Protocols in Pharmacology

Isolate microsomes

5. Pour the homogenate into clean (labeled), open-top 50-ml polycarbonate centrifuge tubes. Balance pairs of tubes using ice-cold microsome preparation buffer.
6. Centrifuge the homogenate for 10 min at 700 × g, 4oC, then increase to 10,000 × g for a further 10 min.Starting at the lower centrifugal force ensures that fibrous tissue sediments first, followed by organelles such as mitochondria. This approach reduces entrapment and improves microsomal protein yield.
7. Carefully remove the supernatant fraction using a syringe with a blunt-end needle and transfer to ice-cold ultracentrifuge tubes (􏰂25 ml). Discard tissue pellets. Balance pairs of tubes using ice-cold microsome preparation buffer.Try to avoid transferring the light-colored fluffy fat layer that ‘floats’ towards the top of the supernatant fraction layer. The supernatant fraction is commonly referred to as the S9 homogenate fraction.
8. Ultracentrifuge the supernatant fraction for 60 min (once maximum speed has been attained 􏰂15 min) at 105,000 × g, 4°C.For example, a Beckman L8-70M ultracentrifuge equipped with a Ti 50.2 rotor is run at 34,000 rpm. Ultracentrifugation sediments the membrane fraction, leaving non- membranous proteins in the supernatant fraction.
9. Decant the supernatant (cytosolic) fraction.
10. Add 3 ml of microsome preparation buffer per g of liver tissue (as determined in step 1) to the ultracentrifuge tube(s).
11. Using a plastic round-ended probe, gently lift the pellet away from the wall of the ultracentrifuge tube(s). Pour the pellet and buffer into a cold 10-ml Potter-Elvehjem grinder.
12. Add an additional 2 ml of microsome preparation buffer to the ultracentrifuge tube(s) and wash away any remaining protein. Combine with the liquid in the Potter-Elvehjem grinder.
13. Resuspend the pellet by gentle hand homogenization (three to four full strokes).
14. Pour the homogenate back into the ultracentrifuge tube(s). Balance pairs of tubes using ice-cold microsome preparation buffer.
15. Ultracentrifuge the homogenate 60 min at 105,000 × g, 4°C, as for step 8.
16. Decant the supernatant fraction and add 1 ml of microsome storage buffer per g ofliver tissue. Repeat step 11 and step 13.
17. Set aside a small portion to determine the protein concentration (APPENDIX 3A; Olson, 2016).
18. Aliquot the remainder of microsomal homogenate into labeled glass vials cooled on dry ice to snap freeze before storing at −70°C.While microsomes are stable for up to 10 freeze-thaw cycles (Pearce et al., 1996), freezing in portions sufficient for a single experiment eliminates multiple freeze/thaw cycles as a source of variability.For proteomic analyses, omit glycerol from the microsome storage buffer, which may affect the stability of CYP enzymes.

http://cshprotocols.cshlp.org/content/2014/8/pdb.prot079970.full

https://currentprotocols.onlinelibrary.wiley.com/doi/10.1002/cpph.9