The potential contribution of CYP3A in brain microsomes and mitochondria is crucial to designing appropriate therapeutic strategies and preventing toxicities. Until recently, there has been limited data reported in the literature with respect to CYP3A activity and its expression in the brain tissue. The reason for this limitation is most likely the relatively low level of CYP3A in the brain tissue. To this end, the primary purpose of this study is to develop a CYP3A functional assay in rat brain mitochondria and microsomes to determine the Michaelis-Menten kinetics of the formation of CYP3A-mediated hydroxylated metabolites of midazolam by UPLC-MS/MS. We also aim to analyze the protein levels of CYP3A in the rat brain mitochondria and microsomes by Western blot analysis.
Microsome and mitochondria were prepared using different centrifugation methods, followed by purification of the crude mitochondrial fractions using the Percoll density gradient method. The purity of the fractions was determined by Western blot analysis of the microsomal (calreticulin) and mitochondrial (VDAC) markers. Different concentrations of midazolam were incubated with rat brain microsomes and mitochondria in a side-by-side comparison over a period of 10 minutes.
The method was developed and optimized to determine the Michaelis-Menten kinetics of MDZ 1′- and 4-hydroxylase activities in rat brain microsomes and mitochondria (n=8). For 4- hydroxymidazolam, brain mitochondria had 2.5-fold higher maximum velocity and 5-fold higher MM constant compared to microsomes. For 1’-hydroxymidazolam, mitochondria had 9-fold higher maximum velocity and 153-fold higher MM constant compared to microsomes. Consistent with the higher Vmax of the metabolites in the mitochondrial fractions, the Western blot analysis showed higher band intensities related to CYP3A2 in the brain mitochondria, when compared with the brain microsomes. Our in vitro study indicates that in the brain the content and activity of CYP3A in the mitochondria are higher than those in the microsomes, an observation which is opposite of the pattern reported in the liver. The local metabolism of CYP3A substrates in the brain microsomes and mitochondria may have significant ramifications for the effectiveness or toxicity of the centrally-acting drugs that are CYP3A substrates.
","abstract_html":"<p>The potential contribution of CYP3A in brain microsomes and mitochondria is crucial to designing appropriate therapeutic strategies and preventing toxicities. Until recently, there has been limited data reported in the literature with respect to CYP3A activity and its expression in the brain tissue. The reason for this limitation is most likely the relatively low level of CYP3A in the brain tissue. To this end, the primary purpose of this study is to develop a CYP3A functional assay in rat brain mitochondria and microsomes to determine the Michaelis-Menten kinetics of the formation of CYP3A-mediated hydroxylated metabolites of midazolam by UPLC-MS/MS. We also aim to analyze the protein levels of CYP3A in the rat brain mitochondria and microsomes by Western blot analysis.</p> <p>Microsome and mitochondria were prepared using different centrifugation methods, followed by purification of the crude mitochondrial fractions using the Percoll density gradient method. The purity of the fractions was determined by Western blot analysis of the microsomal (calreticulin) and mitochondrial (VDAC) markers. Different concentrations of midazolam were incubated with rat brain microsomes and mitochondria in a side-by-side comparison over a period of 10 minutes.</p> <p>The method was developed and optimized to determine the Michaelis-Menten kinetics of MDZ 1′- and 4-hydroxylase activities in rat brain microsomes and mitochondria (n=8). For 4- hydroxymidazolam, brain mitochondria had 2.5-fold higher maximum velocity and 5-fold higher MM constant compared to microsomes. For 1’-hydroxymidazolam, mitochondria had 9-fold higher maximum velocity and 153-fold higher MM constant compared to microsomes. Consistent with the higher Vmax of the metabolites in the mitochondrial fractions, the Western blot analysis showed higher band intensities related to CYP3A2 in the brain mitochondria, when compared with the brain microsomes. Our in vitro study indicates that in the brain the content and activity of CYP3A in the mitochondria are higher than those in the microsomes, an observation which is opposite of the pattern reported in the liver. The local metabolism of CYP3A substrates in the brain microsomes and mitochondria may have significant ramifications for the effectiveness or toxicity of the centrally-acting drugs that are CYP3A substrates.</p>","abstract_has_math":false,"creators":["Alshammari, Nouf"],"institution":null,"degree_name":"Master of Science (MS)","degree_level":"Thesis","degree_discipline":"Pharmaceutical Sciences","degree_department":null,"school":null,"contributors":["Reza Mehvar","Ajay Sharma","Aftab Ahmed"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2022,"date_issued":"2022-05-01T07:00:00Z","date_published":"2022-05-01T07:00:00Z","updated_at":"2026-07-24T01:38:24Z","subjects":["Other Pharmacy and Pharmaceutical Sciences"],"languages":[],"rights":[],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"https://digitalcommons.chapman.edu/pharmaceutical_sciences_theses/21","outbound_label":"Repository record","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Reza Mehvar","Ajay Sharma","Aftab Ahmed"]},{"key":"dc:creator","label":"Author","values":["Alshammari, Nouf"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"thesis:degree_discipline","label":"Discipline","values":["Pharmaceutical Sciences"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Thesis"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Master of Science (MS)"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Other Pharmacy and Pharmaceutical Sciences"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["https://digitalcommons.chapman.edu/pharmaceutical_sciences_theses/21"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["The potential contribution of CYP3A in brain microsomes and mitochondria is crucial to designing appropriate therapeutic strategies and preventing toxicities. Until recently, there has been limited data reported in the literature with respect to CYP3A activity and its expression in the brain tissue. The reason for this limitation is most likely the relatively low level of CYP3A in the brain tissue. To this end, the primary purpose of this study is to develop a CYP3A functional assay in rat brain mitochondria and microsomes to determine the Michaelis-Menten kinetics of the formation of CYP3A-mediated hydroxylated metabolites of midazolam by UPLC-MS/MS. We also aim to analyze the protein levels of CYP3A in the rat brain mitochondria and microsomes by Western blot analysis.
Microsome and mitochondria were prepared using different centrifugation methods, followed by purification of the crude mitochondrial fractions using the Percoll density gradient method. The purity of the fractions was determined by Western blot analysis of the microsomal (calreticulin) and mitochondrial (VDAC) markers. Different concentrations of midazolam were incubated with rat brain microsomes and mitochondria in a side-by-side comparison over a period of 10 minutes.
The method was developed and optimized to determine the Michaelis-Menten kinetics of MDZ 1′- and 4-hydroxylase activities in rat brain microsomes and mitochondria (n=8). For 4- hydroxymidazolam, brain mitochondria had 2.5-fold higher maximum velocity and 5-fold higher MM constant compared to microsomes. For 1’-hydroxymidazolam, mitochondria had 9-fold higher maximum velocity and 153-fold higher MM constant compared to microsomes. Consistent with the higher Vmax of the metabolites in the mitochondrial fractions, the Western blot analysis showed higher band intensities related to CYP3A2 in the brain mitochondria, when compared with the brain microsomes. Our in vitro study indicates that in the brain the content and activity of CYP3A in the mitochondria are higher than those in the microsomes, an observation which is opposite of the pattern reported in the liver. The local metabolism of CYP3A substrates in the brain microsomes and mitochondria may have significant ramifications for the effectiveness or toxicity of the centrally-acting drugs that are CYP3A substrates.
"]},{"key":"dc:source","label":"Dc Source","values":["Alshammari, N. Content and Activity of Cytochrome P450 3A in Rat Brain Microsomes and Mitochondria. [master’s thesis]. Irvine, CA: Chapman University; 2022. https://doi.org/10.36837/chapman.000364"]},{"key":"dc:title","label":"Title","values":["Content and Activity of Cytochrome P450 3A in Rat Brain Microsomes and Mitochondria"]}]}],"canonical_facts":{"dc:contributor":["Reza Mehvar","Ajay Sharma","Aftab Ahmed"],"dc:creator":["Alshammari, Nouf"],"dc:description.abstract":["The potential contribution of CYP3A in brain microsomes and mitochondria is crucial to designing appropriate therapeutic strategies and preventing toxicities. Until recently, there has been limited data reported in the literature with respect to CYP3A activity and its expression in the brain tissue. The reason for this limitation is most likely the relatively low level of CYP3A in the brain tissue. To this end, the primary purpose of this study is to develop a CYP3A functional assay in rat brain mitochondria and microsomes to determine the Michaelis-Menten kinetics of the formation of CYP3A-mediated hydroxylated metabolites of midazolam by UPLC-MS/MS. We also aim to analyze the protein levels of CYP3A in the rat brain mitochondria and microsomes by Western blot analysis.
Microsome and mitochondria were prepared using different centrifugation methods, followed by purification of the crude mitochondrial fractions using the Percoll density gradient method. The purity of the fractions was determined by Western blot analysis of the microsomal (calreticulin) and mitochondrial (VDAC) markers. Different concentrations of midazolam were incubated with rat brain microsomes and mitochondria in a side-by-side comparison over a period of 10 minutes.
The method was developed and optimized to determine the Michaelis-Menten kinetics of MDZ 1′- and 4-hydroxylase activities in rat brain microsomes and mitochondria (n=8). For 4- hydroxymidazolam, brain mitochondria had 2.5-fold higher maximum velocity and 5-fold higher MM constant compared to microsomes. For 1’-hydroxymidazolam, mitochondria had 9-fold higher maximum velocity and 153-fold higher MM constant compared to microsomes. Consistent with the higher Vmax of the metabolites in the mitochondrial fractions, the Western blot analysis showed higher band intensities related to CYP3A2 in the brain mitochondria, when compared with the brain microsomes. Our in vitro study indicates that in the brain the content and activity of CYP3A in the mitochondria are higher than those in the microsomes, an observation which is opposite of the pattern reported in the liver. The local metabolism of CYP3A substrates in the brain microsomes and mitochondria may have significant ramifications for the effectiveness or toxicity of the centrally-acting drugs that are CYP3A substrates.
"],"dc:identifier":["https://digitalcommons.chapman.edu/pharmaceutical_sciences_theses/21"],"dc:source":["Alshammari, N. Content and Activity of Cytochrome P450 3A in Rat Brain Microsomes and Mitochondria. [master’s thesis]. Irvine, CA: Chapman University; 2022. https://doi.org/10.36837/chapman.000364"],"dc:subject":["Other Pharmacy and Pharmaceutical Sciences"],"dc:title":["Content and Activity of Cytochrome P450 3A in Rat Brain Microsomes and Mitochondria"],"thesis:degree_discipline":["Pharmaceutical Sciences"],"thesis:degree_level":["Thesis"],"thesis:degree_name":["Master of Science (MS)"]},"updated_at":"2026-07-24T01:38:24Z"}