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The following message was posted to: PharmPK
I have tried preparing rat intestine microsomes according to the
following methods but obtained only 95ug of protein from 30 cm of
male Sprague-Dawley rat small intestine. The only difference from the
following procedures is that upon obtaining the intestine it was in
solution A for around 20 minutes instead of being immediately
transferred to solution B.
I would appreciate any suggestions or alternative methods for
preparation.
Enterocyte Isolation:
Johnson T.N., Tanner M.S., Tucker G.T. Comparison of the
Ontogeny of Enterocytic and Hepatic Cytochromes P450 3A in the Rat.
Biochemical Pharmacology, Vol. 60, pp. 1601-1610, 2000.
"The bowel sections were
immediately placed in ice-cold buffer (PBS containing 5
mM EDTA, 0.5 mM dithiothreitol, and 40 mg/mL of
phenylmethylsulphonyl fluoride). The bowel contents were
flushed out and the tissue transferred to fresh buffer. A
small cross-section of duodenum (1-2 mm) was taken from
each bowel length and placed in buffered formaldehyde for
histochemical staining. The remaining bowel samples were
dissected longitudinally, chopped into 2- to 3-cm lengths
and placed in 100 mL ice-cold modified Weiser solution
[19]. After stirring gently for 15 min the enterocytecontaining
suspension was decanted into a glass beaker on
ice. This process was repeated with 100 mL of fresh ice-cold
Weiser solution. The two Weiser fractions were then
combined and centrifuged in 50-mL aliquots at 800 g for 10
min. The cell pellets were then combined, re-suspended,
and washed twice with 10 mL of buffer (histidine 5 mM,
sucrose 0.25 M, EDTA 0.5 mM, and phenylmethylsulphonyl
fluoride 40 mg/mL; pH 7.4), and centrifuged at 800 g for 10 min."
Microsomal Preparation:
2. Cotreaua M.M., von Moltke L.L., Beinfelda M.C., Greenblatt D.J.
Methodologies to study the induction of rat hepatic and intestinal
cytochrome P450 3A at the mRNA, protein, and catalytic activity
level. Journal of Pharmacological and Toxicological Methods 43 (2000)
41-54
" At all points in the microsomal
preparation the tissue was maintained at 4*C. All buffers,
except the final resuspension buffer (phosphate buffer and
glycerol), contained PMSF at 40 mg/ml, which was added
immediately prior to the use of each solution. Intestinal
microsomes were prepared for each rat independently,
there was no pooling of tissue from different rats. The
intestinal pellets were each resuspended in 5 ml of solution
C and homogenized in a loose-fitting Dounce homogenizer.
The homogenates were placed into clean centrifuge
tubes. The homogenizer was washed with an additional 2
ml of solution C and this was added to the initial
homogenate. The total homogenate was then centrifuged
at 15,000 g for 10 min. The supernatant was carefully
transferred to a clean centrifuge tube. The pellet was
resuspended in 5 ml fresh solution C and centrifuged
again at 15,000 g for 10 min. The supernatant was
carefully removed and added to the first supernatant
fraction. For each 7 ml of supernatant collected, 1.25 ml
of 52 mM CaCl2 was added. Tubes were gently shaken for
5 s and then allowed to stand for 15 min. Fractions were
then centrifuged at 25,000 g for 10 min."
Maggie Abbassi
Graduate Student
Pharmaceutical Sciences
University of Kentucky Lexington, KY
mabba2.-at-.email.uky.edu
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The following message was posted to: PharmPK
Hi Maggie,
we use Dounce homogenizer to obtain intact mitochondria from liver. We
stroke 8 fold with loose pestle and 4 fold, very gently, with tight
pestle. Since mitos are bigger and more fragile than microsomes you can
use the tight pestle and obtain a better homogenate.Be sure to mantain
pestles and Dounce always in melting ice since different temperatures
could result in different interspace between pestle and tube.
In the past, I prepared liver and brain microsomes by tissue
homogenation (1gr in 10ml of buffer) and subsequent centrifugations in
buffered sucrose 0.25M. The first centrifugation is to be done at 20000g
x 10' and the resulting supernatant at 100000g x 60'.
Since I don't think that liver or brain microsomes are different from
the intestine one, in the resulting pellet you'll find microsomes from
intestine.
Ciao Roberto
Dr Roberto Conti
Sigma-Tau
Dep. of Endocrinology and Metabolism
00040 pomezia, Roma
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The following message was posted to: PharmPK
Maggie,
I don't have any experience with either of the two methods you
describe (non-enymatic release of enterocytes, microsomal fraction
by calcium aggregation). I understand that the former is primarily
intended to provide relatively pure enterocytes for mRNA or immunoblot
analysis but the yield is unlikely to be high enough for large-scale
enzymological analyses (unless optimised in some way). I have never
had any luck with any method that involves homogenisation of the
entire intestine as the relevant cell population (villus tip
enterocytes) becomes too diluted with those in the underlying
connective tissue and muscular layers.
The method I have used is the "scrape" preparation. The intestine
is flushed with ice-cold buffer and then slit longitudinally. It
is then placed, lumen side up, on a flat surface (e.g. a chilled
glass plate) and the surface layer scraped away with the edge of
a microscope slide. The resulting material is then diluted with
buffer, homogenised, spun at 9,000 x g, and the supernatant spun
at 105,000 x g, as normal. As noted in the two methods that you
quote PMSF (or AEBSF etc.) is essential and DTT is helpful (some
people also add heparin but I have found no improvement with this).
It is also helpful to have EDTA (e.g. 1 mM) in the homogenisation
medium. You can obtain microsomal fractions that give reasonable
CO difference spectra by this method.
One of the main problems is normalising the yield of microsomal
fraction as the concentration of total protein is influenced by
the level of contamination by non-enterocytes (scraping harder will
increase the yield somewhat but will reduce the purity drastically).
One method I have seen used is to determine the concentration of
villin by immunoblot analysis and normalise activities to this.
I do not have wide experience with preparation of intestinal
fractions but I hope that this gives you some ideas.
All the very best,
Bernard
Bernard Murray, Ph.D.
Senior Research Scientist, Drug Metabolism
Gilead Sciences
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The following message was posted to: PharmPK
Dr. Murray,
Thanks a lot for the response. My work requires normalizing to protein
content in general, where villin only would not be adequate. This is why
I am not comfortable using the scraping method. I am trying to find a
different method or modification to the methods I have described where
normalizing to total protein would be possible.
Thanks,
Maggie
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