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The following message was posted to: PharmPK
Hello,
We are trying to account for the low recovery of an
i.v. dosed radiolabeled compound. Does anyone have
advice or experience on homogenizing whole rats.
Thanks,
Stephen Day
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(I am assuming you are not losing the label as [14C]carbon dioxide or
[3H]water) Rather than homogenising the entire carcasse I would suggest
pulling representative tissues and checking them to give you some idea
of
where the label is hanging up. If it is fairly localised this would
give
a strong signal and make quantification easier. It would also make it
easier for you to check for covalent attachment or perhaps even allow
extraction for a radiochemical profile.
All the very best,
Bernard
Bernard Murray, Ph.D.
Drug Metabolism, PCS, PPD, GPRD, Abbott Laboratories, Chicago, USA
Bernard.Murray.aaa.abbott.com
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[Three more replies - db]
From: "James Liddil"
Date: Tue Feb 18, 2003 2:30:03 PM US/Central
To: david.-a-.boomer.org
Subject: RE: PharmPK Whole rat homogenization
Reply-To: "James Liddil"
The following message was posted to: PharmPK
You need the Ronco Bass-o-matic. (Old SNL skit) But seriously I would
think
you could use a blender of the industrial variety. Passivate the parts
and
try to see what can be done to prevent non-specific binding. Or it the
radioactive element is non-volative get a large Teflon Bomb and reduce
it to
ash.
Jim Liddil MS
Manager of Information and Technology Services
Phytoceutica Inc.
203-781-2544
---
From: "Daniel Sitar"
Date: Tue Feb 18, 2003 2:43:57 PM US/Central
To: david.at.boomer.org
Subject: Re: PharmPK Whole rat homogenization
Reply-To: sitar.-at-.Ms.UManitoba.CA
We used to digest the carcases in hot sodium
hydroxide solution.
Dan Sitar, University of Manitoba
---
From: tongw.-at-.mskcc.org
Date: Tue Feb 18, 2003 2:41:44 PM US/Central
To: david.aaa.boomer.org
Subject: Re: PharmPK Whole rat homogenization
Reply-To: tongw.at.mskcc.org
The following message was posted to: PharmPK
Stephen,
We used about 1 liter of ~50% potassium hydroxide/ethanol(water?)
in a
1 liter fleaker. After all the organs are removed, the remain is
put
into this solution. Just let it sit and it will dissoved in about
2-4
weeks than just take a sample and count. Teeth are the last to
dissolve.
Bill Tong
Memorial Sloan Kettering Cancer Center
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The following message was posted to: PharmPK
A way they used at Cyanamid was to take the frozen remains and pass them
repeatedly through a Hobart industrial meat grinder with dry ice, then
store
the powder in a freezer until it reached constant weight.
Appropriate mixing would yield a homogeneous sample which could be
digested
and counted...
Bill O'Neil
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[Three replies - db]
From: Hans-Markus.Bender.-a-.merck.de
Date: Wed Feb 19, 2003 1:02:46 AM US/Central
To: david.-a-.boomer.org
Subject: Antwort: PharmPK Whole rat homogenization
Reply-To: Hans-Markus.Bender.aaa.merck.de
The following message was posted to: PharmPK
Hallo Stephen,
we usually determine the total 14C-radioactivity in the residual body by
the method described below. In iv dosed rats the tail, which is the iv
injection site in our lab, is separately measured to check proper
injection. In mass balance or organ distribution studies we usally get
recoveries of 100% plus / minus 3%, if 14CO2 formation is negligible.
To determine the radioactivity remaining in the organism after
removal
of all organs and tissue samples the remaining parts of the
animal
bodies were weighed, frozen and thoroughly minced with an
electrical
mincer. Three 0.5 g samples of the homogenate were weighed
into
Combusto-Cones. To determine the radioactivity remaining at
the
in-jection site, the tails of the rats used in the iv experiments
were
removed prior to homogenization, cut into 20-30 segments and
filled
into Combusto-Cones. Sample oxidation: All samples were dried
in an
oven at 60°C for 12 h, pressed with a tablet press
(PAAR-Instruments,
USA) and oxidized in a PACKARD TriCarb 307 sample oxidizer. 7
ml of
2-methoxyethylamine (BASF, Ludwigshafen) was used as the
CO2
absorbent; the volume was subsequently made up with 13
ml
Omni-Szintisol (MERCK Cat.No. 15 386). Measurement by LSC ...
Regards
Markus
Dr. Hans Markus Bender
Merck KGaA
Institute of Drug Metabolism and Pharmacokinetics
Am Feld 32
D-85567 Grafing
[Germany]
---
From: "Zutshi, Anup [R&D/0216]"
Date: Wed Feb 19, 2003 8:21:06 AM US/Central
To: david.aaa.boomer.org
Subject: RE: PharmPK Re: Whole rat homogenization
Reply-To: "Zutshi, Anup [R&D/0216]"
The following message was posted to: PharmPK
Stephen
You can do this two ways. Both have been suggested to you in different
responses.
1) Physical homogenization using a `Waring' blender (industrial
strength as suggested by some). The problem with this method is that
skin
(and fur) and some bones do not get homogenized very well (infact large
chunks of skin tissue will wrap around the rotor blades and freeze it)
and by the time you get it done to your satisfaction you will have
created
a messy situation.
2) Chemical homogenization consists of immersing the rodent (euthanized
at this point---hopefully (;~))in 2 volume equivalents of its terminal
weight (i.e. a 100g rat will require at least 200 mL) of a homogenizing
solution. The homogenizing solution is a mixture of 8M Potassium
hydroxide
in water (500mL) and 4M Benzalkonium Chloride in Methanol (500 mL).
The two solutions are mixed and can be stored for approximately 3
months in
air-tight containers. If done at room temperature it takes about 2
weeks for the carcass to be solubilized. If you hook-up a soxhelet
condenser
(or any water cooled condenser will do) and heat the contents of the
flask on a heating mantle to just about a gentle boil, the entire
process can be accomplished in ~16 hours (or overnight). The homogenate
can
be sampled as a liquid (reddish brown in color and somewhat foul
smelling) and processed for counting. If you plan to oxidize the sample
in a
Packard Oxidizer make sure that you have neutralized your aliquot
(with concentrated acid) before oxidizing it. If directly counting
(after decolorizing) use a cocktail that can count samples of high ionic
strength (Packards Hionic-FluorTM).
Good Luck
Anup Zutshi Ph.D.
Global Drug Metabolism
Pharmacia
Mail Stop: 7260-300-104.1
Kalamazoo, MI 49001
Tel: (269) 833-8347
Fax: (269) 833-7788
Anup.Zutshi.aaa.Pharmacia.com
---
From: "Tata, Prasad N"
Date: Wed Feb 19, 2003 8:59:47 AM US/Central
To: david.at.boomer.org
Subject: RE: PharmPK Whole rat homogenization
Reply-To: "Tata, Prasad N"
The following message was posted to: PharmPK
Stephen:
More elaborate question would have helped us to offer constructive
suggestion. The way I understood your question was you are trying to do
a
mass balance study after an IV administration.
This can be accomplished by the following steps.
1. Real time radio - imaging if your drug can be radio imaged (one
offered
by Pharmaceutical Profiles).
2. Collecting urine, feces and exhaled air and counting radioactivity
if you
account for 90% of the dose it is considered mass-balance is
accomplished.
3. if you are doing a PK study then whole body autoradiography with
cryoblock may be an answer.
4. Whole rat homogenization is an option but have several practical
issues,
way back Corning-Hazleton in UK were doing digestion of whole animal in
very
strong alkali (50% sodium hydroxide), when I inquired what was the
purpose,
I got an answer that this is to test pesticide and environmental
residues in
food producing animals. By digesting you will get a homogenous mass and
measurements are more reliable.
Hope this info is useful, my personal preference would be 1, 2, and 3 in
that order but not 4.
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The following message was posted to: PharmPK
Dear Stephen,
If you used [14C] and no cardiac puncture in your
experiment you could also use quantitative whole body
autoradioluminography (QWBA). It is kind of expensive
but I assume one rat would be enough for
investigational purposes.
Rostam
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The following message was posted to: PharmPK
Some time ago, I was pulverizing small rats by dipping them whole in
liquid nitrogen (5 min), and then smashing them in custom machined
stainless steel mortar cooled in liquid N2, using loosely fitted pestle
and hammer. This method worked even for semivolatile chemicals. You can
extract it whole (be sure to weight it when still stiff frozen) in
known volume of medium (calculated per g of tissue).
I hope, it may help.
Janusz Z. Byczkowski, Ph.D.,D.Sc.,D.A.B.T.
Consultant
212 N. Central Ave.
Fairborn, OH 45324
voice (937)878-5531
e-mail januszb.-at-.AOL.com
homepage: http://members.aol.com/JanuszB/index.html
JZB Consulting web site: http://members.aol.com/JanuszB/consult.htm
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