Human and rat liver microsomes are subcellular fractions containing cytochrome P450 enzymes and other drug-metabolizing enzymes. They are commonly used in vitro to study drug metabolism and interactions. Human liver microsomes are obtained from human liver tissue through differential centrifugation and contain enzymes for phase I and phase II drug metabolism. They are useful for identifying drug metabolites, evaluating interspecies differences, predicting in vivo clearance, and studying interindividual variability in drug metabolism. Rat liver microsomes were also discussed as an experimental model. Incubation methods and analytical techniques like HPLC were described for evaluating metabolism using liver microsomes.

1. Human Liver Microsomes & Rat Liver Microsomes
PRESENTED BY - GAURAV SHARMA
M.PHARM (2nd sem.)
P’CEUTICAL ANALYSIS

2. Human liver microsomes
(HLM)
• HLM are vesicles of hepatocyte endoplasmic reticulum.
• It is obtained by differential centrifugation of liver preparations
(homogenates) from fresh human liver, liver slices, liver cell lines and
primary hepatocytes.
• It is a rich source of following enzymes such as:
• cytochrome P450s,
• flavin- monooxigenase (FMO),
• carboxyl esterases , epoxide
• hydrolase and UGTs (Uridine 5-diphospho-glucuronosyltransferase).

3. • Therefore, HLM are most frequently utilized in vitro model in drug
metabolic profiling and drug interaction studies.
• The influence of specific isoenzymes is studied using liver microsomes
in the presence of specific inhibitors.
• There are inter individual variations in the activity of human liver
microsomes therefore they can be utilized also to study inter
individual variability.
• In case of estimation of drug metabolism, pooled microsomes from a
large bank of individual liver tissues can be used to overcome the
influence of inter individual variability.
• Microsomes from other human organs (intestine, kidney, lung) are
also available and are utilized to evaluate extrahepatic metabolism.

4. • Additionally, gender-specific microsomes are available for the
estimation of gender-based discrepancies in drug
biotransformation.
• In drug discovery process HLM are used for :
 Metabolite identification
 Evaluation of interspecies differences in drug metabolism
 Prediction of in vivo clearance
 Reaction phenotyping and metabolic pathway identification
NADPH(nicotinamide dinucleotide phosphate) or NRS is required
in the incubation for the estimation of CYP activity. In order to
evaluate the UGT activity UDPGA( uridine 5-diphosphoglucuronic
acid) and alamethicin (pore-forming reagent) are required .

5. Advantages of HLM
 Ease of use.
 Low costs.
 Best-characterized in vitro model for estimation of drug
biotransformation.
 Easy storage.
 Appropriate for studying of inter individual and population-
based variation.
 Long term storage.
 Provide qualitative estimations of in vitro drug metabolism.
 Convenient tool for high throughput screening of compounds.
 Appropriate for lead compound optimization studies and drug
interaction studies.

6. Disadvantages of HLM
• HLM are not appropriate for quantitative estimation of drug
biotransformation because of absence of enzymes like NAT(N-
acetyltransferase), GST(Glutathione S-transferase) and
SULT(sulfotransferase) and cofactors needed.
• Another drawback is a very difficult assessment of the fraction
of drug bound to plasma proteins versus to microsomes which
is an important factor in the estimation of in vivo
biotransformation.

7. Liver Microsome Preparation Protocol
Gloves
• Lab Coat
• Pipettors
• Pipette Tips
• Ice Bucket
• Water
• DEPC Water (diethyl pyrocarbonate)
• Eppendorf Tubes
• Pestle Motor

8. Pestle Motor
• Pestles
• EDTA
• Sucrose
• APMSF (4 Amidinophenylmethylsulfonyl fluoride
hydrochloride
• Glycerol
• KH2PO4

9. Prepare Homogenization Buffer. Combine 320mM sucrose,
50mM KH2PO4, 1mM EDTA, and 1mM APMSF and adjust pH
to 7.40.
• Prepare Freezing Buffer. Combine 100mM PKi, 1mM EDTA,
1mM APMSF, and 20% (v/v) glycerol and adjust pH to 7.40.
Homogenize Samples .
Thaw tissue samples.
• Blot the liver tissue with paper towels to remove moisture.
• Trim away connective tissue.
• Weigh and record the wet tissue weight.
• Add homogenization buffer to the tissue (500 uL buffer per
g tissue).

10. • Homogenize the tissue samples using motor-driven pestle.
• Add additional homogenization buffer to the tissue samples (1.5
mL buffer per g tissue).
• Vortex for 30 s.
• Centrifuge samples @ 10,000G, 10 min, 4C.
• Transfer the supernatant to a new Eppendorf tube.
• Centrifuge samples @ max speed, 20 min, 4C.
• Discard the supernatant.
• Wash the pellet gently with homogenization buffer. Discard the
excess homogenization buffer.
• Resuspended the pellet in freezing buffer (500 uL buffer per g
tissue) with gentle pipetting to break up the pellet.

11. • Measure absorbance of DNA using nanodrop spectrometer.
• Blank spectrometer with 1 uL DEPC water.
• Load 1 uL DNA samples onto spectrometer, recording 260/280 and
260/230 values.
• Make duplicate measurements of all samples. If inconsistent, make
extra measurements as necessary.
• For best results, resuspend DNA pellet before measuring
absorbance (gently do this by flicking the tube several times with
your finger).

12. Rat Liver Microsomes
• Livers were harvested from Wistar and brown rats
(Rattus norvegicus) and bobwhite quail (Colinus
virginianus).
• Following euthanasia by CO2, a longitudinal incision was
made in the abdomen, and the liver was removed and
weighed.
• The tissue was rinsed thoroughly
• Then perfused with ice-cold 0.9% NaCl or immediately
frozen in liquid nitrogen.

13. • The perfusion was continued until the liver appeared blanched.
• The perfused tissue was then snap-frozen in liquid nitrogen.
• The frozen samples were stored at -80°C until further processing.
Test Solutions
• All test formulations consisted of diphacinone or chlorophacinone
solutions at approximately 40 ppm in phosphate (0.010 M) solutions
buffered at a pH of7.4.
• Solutions of magnesium chloride, phosphate buffer, and NADH were
all 0.010 M.
• Homogenization buffer was as follows: sucrose 250 mM, KCl 25 mM,
MgCl2 5 mM, EDTA 0.1 mM, adjust pH to 7.4.

14. • Cofactor solution was made by adding 11.5 mg NADP sodium salt,
5.2 mg glucose-6-phosphate, and 50 μL glucose-6-phosphate
dehydrogenase to 950 μL 0.01 M MgCl in phosphate buffer
• Liver Microsome Preparation
• Livers microsomes were isolated using differential centrifugation
according to the method by Pelkonen et al. (1974) with minor
alterations.
• Frozen liver samples were minced,
• weighed, and transferred to a Teflon pestle/glass homogenizer with
2 volumes (w/v) homogenization buffer with the addition of phenyl
methanesulfonyl fluoride 2.5 μL/ mL homogenization buffer.

15. • The tissue was homogenized with 6 passes of the Teflon pestle
homogenizer.
• The homogenates were centrifuged at 10,000 g for 10 min at 4°C.
• The supernatants were transferred to a clean centrifuge tube and
spun at 15,000 g for 20 minutes at 4°C.
• The supernatant was then transferred to ultracentrifuge tubes and
spun at 105,000 g for 60 minutes at 4°C.
• The pellets were then washed with approximately 1 mL
homogenization buffer.
• Transferred to the Teflon pestle/glass homogenizer and
resuspended in homogenization buffer and spun at 105,000 g for
60 minutes at 4°C

16. • The supernatants were discarded, and the remaining pellets were
resuspended in homogenization buffer using Teflon pestle/glass
homogenizer and frozen at -80°C.
• Microsome Incubation
• Microsome incubations were performed using 50 μL microsome
extract, 50 μL cofactor solution, the analyte, and 0.01 M phosphate
buffer added to bring volume to 500 μL.
• These incubations contained 2.4 ppm of either diphacinone or
chlorophacinone.
• Incubations were done at 37°C for 60 minutes.

17. Residue Determination
• Chlorophacinone and diphacinone residue determination was
completed by quenching 0.400 mL of the incubation solution with
0.600 mL of methanol containing 5 mM tetrabutylammonium
phosphate and vortex mixing.
• These samples are filtered with 0.45 μm Teflon syringe.
• Filters prior to analysis by reverse phase ion-pairing high
performance liquid chromatography (HPLC) using the following
parameters:
55:45 methanol:water w/5 mM tetrabutylammonium phosphate, 10
mM phosphate buffer pH = 8.5.

18. • octadecyl silane column 150 mm × 3.0 mm & particle size
3 μm at 0.300 mL/min at 35°C.
• UV detection at 325 nm with UV spectral confirmation
using an Agilient 1100 with ChemStation software.