Laboratory Experiment pubs.acs.org/jchemeduc
A P450 Metabolism Experiment for Undergraduate Biochemistry Laboratories Reza Razeghifard,* Catherine E. Chiafair, and Dimitrios G. Giarikos* Division of Math, Science & Technology, Farquhar College of Arts & Sciences, Nova Southeastern University, Fort Lauderdale, Florida 33314, United States S Supporting Information *
ABSTRACT: A laboratory experiment is described to provide students hands-on experience in learning some aspects of microsomal P450-catalyzed metabolism. Undergraduate students in the biochemistry laboratories detect and quantify the metabolites produced from butylated hydroxytoluene (BHT) by liver microsomes using gas chromatography−mass spectrometry (GC−MS) techniques. The laboratory provides training to students for handling active microsomes, sample cleanup of biological matrices, extraction of organic metabolites, GC−MS analysis, data interpretation of complex mixtures, and collaborative work.
KEYWORDS: Upper-Division Undergraduate, Biochemistry, Laboratory Instruction, Collaborative/Cooperative Learning, Bioorganic Chemistry, Qualitative Analysis, Enzymes, Gas Chromatography, Mass Spectrometry, Quantitative Analysis
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Scheme 1. Four-Step Catalytic Mechanism for P450. (Adapted with permission from Davydov et. al.7 Copyright 2008, American Chemical Society)
n important objective of chemistry laboratories is to teach students real-world application of topics learned in lectures. In this paper, an exciting stand-alone experiment involving cytochrome P450 enzyme is described for undergraduate biochemistry laboratories to inspire active learning in biochemistry among students. P450 enzymes are a ubiquitous superfamily of multifunctional oxidases with important catalytic roles including the metabolism of xenobiotics, such as drugs and environmental pollutants, and biosynthesis of steroids and fat-soluble vitamins.1 P450 metabolism can be studied by reacting any of these xenobiotics with microsomal fractions obtained from liver tissues. The versatile reactivity of P450 enzyme toward so many structurally different compounds is believed to be due to its plastic active site.2 Some structures have now become available showing drug interactions with human P450 enzymes, for example, P450 3A4 with ketoconazole and erythromycin,3 P450 2D6 with prinomastat,4 and P450 17A1 and P450 46A1 with prostate cancer drugs abiraterone5 and bicalutamide.6 The catalytic mechanism for P450 is typically described by four steps based on the state of the heme iron and oxygen as shown in Scheme 1.7 It is the reduction of the heme iron−O2 complex by the P450 reductase enzyme using NADPH that starts the reaction cycle by forming the hydroperoxo species and eventually reactive ferryl-oxo πcation porphyrin radical complex, referred to as compound I. There are three interesting aspects to this metabolism experiment that make it suitable for an undergraduate biochemistry laboratory: (a) it is practical because it can be performed using active liver microsomes either prepared by ultracentrifugation8 or purchased from commercial sources, (b) © 2013 American Chemical Society and Division of Chemical Education, Inc.
there are many different compounds from which to choose and each can give unique metabolites when reacted with P450 enzymes, and (c) many of these metabolites can be detected by gas chromatography−mass spectrometry (GC−MS).9−11 GC− MS is a desirable technique for instructional laboratories because multiple student samples can be analyzed in a relatively Published: November 22, 2013 141
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understand biochemistry of drug metabolism taught in the classroom by providing them with a collaborative laboratory experience. Specific learning objectives of this experiment are for students to learn how to (i) handle active microsomes, (ii) extract organic metabolites, (iii) perform sample cleanup of biological matrices, (iv) analyze and interpret GC−MS data of complex mixtures, and (v) work collaboratively.
short time. The technique is well suited for analyzing complex mixtures because the compounds in the mixture are separated through chromatography while being identified and quantified by a sensitive mass spectrometer.12 The experiment was successfully completed by thirty-two students in two laboratory sessions scheduled for three hours each. The students worked in pairs to allow peer interactions and also were facilitated by the instructor and a laboratory assistant. Peer learning has been shown to help teach chemistry to science students.13−15 The instructor guided students so that results were obtained in the time allocated.
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EXPERIMENTAL PROCEDURES
First Laboratory Session
GC−MS conditions for the separation of BHT and its metabolites are optimized by the instructors to allow their detection and quantification. Using this method, students constructed calibration graphs by preparing standards for BHT, BHT-OH, and BHT-CHO. To construct the calibration graphs, the integrated area for each GC−MS peak is calculated and plotted versus concentration. A linear fit to the data points, forced through the origin, is applied to each calibration graph to obtain the fit equation.
OVERVIEW OF THE EXPERIMENT The activity of liver P450 enzymes is tested with 2,6-di-tertbutyl-4-methylphenol (BHT) as the substrate and its metabolites are extracted, detected, and quantified by GC− MS. BHT, which is a widely used food antioxidant, was chosen as a safe alternative compound to drugs. It was previously shown that BHT was metabolized by liver microsomes from rats and mice.16 The two main metabolites produced by P450 are 3,5-di-tert-butyl-4-hydroxybenzyl alcohol (BHT-OH) and 3,5-di-tert-butyl-4-hydroxybenzaldehyde (BHT-CHO) as shown in Figure 1.7 The pedagogical goals are for students to
Second Laboratory Session
Students followed the detailed procedure provided in Scheme 2. The BHT metabolism by P450 is measured by quantifying the quantities of BHT-OH and BHT-CHO metabolites and unreacted BHT using the fit equations obtained from the calibration graphs. The reaction is initiated by providing a continuous supply of NADPH to microsomes using a regenerating system containing NADP+, glucose 6-phosphate, and the glucose 6-phosphate dehydrogenase enzyme. Enough metabolites are generated when microsomes were incubated with BHT for 1 h at 37 °C. BHT and its metabolites are extracted using organic solvents and quantified by GC−MS after sample cleanup.
Figure 1. Structures of 2,6-di-tert-butyl-4-methylphenol (BHT), 3,5-ditert-butyl-4-hydroxybenzyl alcohol (BHT-OH), and 3,5-di-tert-butyl-4hydroxybenzaldehyde (BHT-CHO).
Scheme 2. Procedure for the Sample Cleanup and Extraction of BHT and Its Metabolites from Microsome−BHT Reaction Mixtures
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HAZARDS AND DISPOSAL Protective clothing, gloves, and eyewear must be used in the lab. All of the chemicals used in this laboratory are considered potential irritants to the eyes, skin, and respiratory tract, and most can be toxic if ingested. Avoid inhaling any dust or fumes. Organic solvents such as methylene chloride, methanol, and hexane are flammable and must be kept away from heat, sparks, and open flames. The organic solvents must be handled in the fume hood. Methylene chloride is a potential carcinogen and hexane is a teratogen and a neurotoxin. Nicotinamide is an eye, skin, and respiratory irritant. Pentobarbital is a controlled substance, an irritant, and may cause birth defects. Large quantities released in aquatic and terrestrial environments may have adverse effects. The following chemicals have target organ effects: glycerol, NADP+, magnesium chloride, methylene chloride, methanol, and hexane. 2,6-Di-tert-butyl-4-methylphenol, 3,5-di-tert-butyl-4-hydroxybenzaldehyde hemihydrate, and 3,5-di-tert-butyl-4-hydroxybenzyl alcohol are marine pollutants. More detailed information on hazards and safe handling practices are available on material safety data sheets (MSDS). All used chemicals, including reagents and prepared samples, must be collected in appropriately labeled waste containers for safe chemical and biological disposal.
Course evaluations, comments, student interactions, and a survey showed that the new laboratory experience was exciting as well as challenging for students. The pedagogical goals for student learning in biochemistry were met by assessing their written lab reports, the final ACS biochemistry exam grades, and group discussions.
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CONCLUSIONS A undergraduate biochemistry course is required by many professional and health sciences graduate programs; therefore, analytical reasoning, critical thinking, and problem-solving skills learned through this laboratory are readily transferable to other courses. In addition, this experiment can be adapted and expanded successfully by including other classes of compounds such as drugs, environmental organic pollutants, steroids, and fat-soluble vitamins, as they can also be metabolized by P450 enzymes. The pedagogical goals were met by assessing students’ laboratory reports and their peer-to-peer discussions. By performing this experiment, undergraduate students were exposed to cutting-edge technologies and techniques needed for future research participation at the undergraduate and graduate levels and for careers in medicine, biotechnology, and the pharmaceutical industry.
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RESULTS An example of the GC−MS data obtained by a pair of students for the BHT metabolism by rat microsomal P450 is shown in Figure 2. The GC−MS data showed that the 4-hydroxymethyl
ASSOCIATED CONTENT
S Supporting Information *
Instructor notes; student handout; detailed information of GCMS settings; mass spectra; calibration graphs; materials; equipment; hazards and disposal. This material is available via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Authors
*R. Razeghifard. E-mail:
[email protected]. *D. G. Giarkios. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We would like to acknowledge the contribution of the class of 2012−2013 biochemistry students in conducting these experiments. This work was in part supported by a NSU President’s Faculty Research and Development Grant.
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Figure 2. Example of GC−MS data obtained by a pair of students for the BHT metabolism using rat microsomes. BHT, BHT-CHO, and BHT-OH are labeled as peaks 1−3, respectively. The other labeled major peaks are nicotinamide* and pentobarbital** (extracted from microsomes), respectively. Nicotinamide originated from NADP+ present in the aqueous layer due to poor extraction techniques by students.
REFERENCES
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