Hydrolytic drug-metabolizing enzymes (hydrolases) are essential for the metabolism of many therapeutic agents; however, comprehensive data on their tissue distribution and interspecies variability remain limited. To address this knowledge gap, the primary objective of this study was to quantify the abundance of drug metabolism-relevant hydrolases (e.g., CES1/2, PON1/2/3, BPHL, APEH, CMBL, EPHX1/2, DPP4, and AADAC) across liver, intestine, and kidney tissues in humans, rats, mice, dogs, and monkeys using a comprehensive global proteomics approach. Further, we present here qualitative and quantitative differences in intertissue and interspecies variability among 182 detected hydrolases. Humans exhibited the greatest variability of hydrolases across tissues, with marked qualitative and quantitative differences in protein abundance observed between species. Orthology analysis highlighted substantial sequence conservation in monkeys but greater divergence in rodents and dogs. Overall, these findings could provide critical quantitative data to inform animal model selection and improve the translation of preclinical drug metabolism studies to humans for drugs that are majorly metabolized by hydrolases.

Uridine 5'-diphospho-glucuronosyltransferases (UGT) are the best known and most well characterized sugar conjugation biotransformation enzymes. Their main function is to catalyze conjugation of glucuronic acid (sugar) to an exposed (or created) hydroxyl, amine or other nucleophilic functional group on a substrate, known as an aglycone. Since these enzymes are subject to inhibition and induction, there is considerable interest in characterizing their role in mediating drug-drug interactions. Glucuronidation reactions are readily recreated in vitro, although most researchers agree rates of catalysis continue to be underestimated compared to those in vivo, thus hampering the prediction of human systemic clearance. Similarly, the magnitude of UGT induction observed in vitro appears to be smaller than those reported in clinical trials. Despite these shortcomings, drug candidates and marketed drugs rarely encounter clinically relevant pharmacokinetic changes attributable to UGT enzyme inhibition or induction. This is likely due to a combination of enzyme properties (high capacity/low affinity, high promiscuity, and minimal inducibility). By contrast, genetic polymorphisms resulting in poor metabolizer or null phenotypes have been demonstrated to have considerable impact on drug disposition, underscoring the need to characterize the fraction of drug metabolized by polymorphic enzymes, especially when glucuronidation is a major clearance mechanism. Recent developments in identifying specific chemical inhibitors and substrate probes, as well as enzyme induction test systems, are helping to further investigate UGT function and role in drug disposition. The aim of this review is to provide an overview of these recent advances and an industry perspective on in vitro testing of UGT-mediated drug-drug interaction potential.

Inhibition of drug-metabolizing enzymes such as P450s and uridine 5'-diphosphoglucuronosyltransferase is routinely evaluated in drug discovery and development. Substantial efforts have been made over the years to standardize in vitro assay conditions and data interpretation. More recently, increasing importance has been given to the approaches for improving translation of in vitro data to predict in vivo outcomes. The International Consortium for Innovation and Quality in Pharmaceutical Development (IQ) Enzyme Inhibition Working Group conducted a survey across IQ member companies to interrogate on the current state of inhibition assessment in the industry, including the strategies used as compounds progress through the pipeline, the kinetic endpoints utilized, and the accuracy of clinical drug-drug interaction predictions. Focus was also placed on how companies applied correction for unbound fraction of potential inhibitors as required by the various recent regulatory guidance documents. Results showed that most companies follow similar set-ups to identify strong inhibitors in discovery and to fully characterize drug candidates in development. Although there are some minor differences in evaluating and correcting for incubational binding, there is almost universal alignment regarding the use of kinetic endpoints with Ki = IC50/2 widely accepted to determine the inhibition constant. A majority of predictions align with clinical drug-drug interaction results, although instances of over- or underprediction were reported, and examples of these are discussed here as case studies. SIGNIFICANCE STATEMENT: Practices utilized to assess reversible inhibition of drug-metabolizing enzymes were evaluated based on 25 survey responses from IQ member companies. Results show how assay conditions, endpoints, and modeling approaches evolve as compounds advance through discovery to development. The accuracy of drug-drug interaction prediction is discussed, and recommendations for best practices are provided.

Microphysiological systems (MPS) are envisioned to improve drug approval success rates. Yet, integration of MPS into drug development processes has been hampered in part by uncertainties in data translation. To speed adoption, development of animal cell-based MPS is advocated by the pharmaceutical industry. In our view, animal MPS availability would fill a key gap in the ability to examine in vitro to in vivo translatability. Since in vivo animal data will be available and guide decision making in regulatory activities for the foreseeable future, there is significant opportunity for translational assessments. In vivo animal study findings that are recapitulated using in vitro models generated from the corresponding animal species provide validation that those models possess the relevant and necessary attributes, e.g., species-specific pharmacodynamics, metabolism, transport, susceptibility to toxicity, etc. Results from the corresponding human models can then be interpreted with greater confidence for the relevant context of use (COU). Some drugs do not get to the clinic due to adverse findings in animals, so there is considerably more data to directly compare to animal in vitro models than human systems. Another benefit of animal MPS is that drug candidates exhibiting animal safety findings might be easier to derisk, for example if the finding was observed in animal but not the corresponding human in vitro model. This paper reviews considerations and recommendations for adopting animal MPS models in drug discovery and development and describes how their deployment is consistent with 3Rs principles.

Microphysiological systems (MPS) contain multiple cell types in three dimensions and often incorporate fluidic shear forces. There is interest in MPS for disease and efficacy modeling, safety and disposition studies. Animal cell-based MPS are needed to provide confidence in the translation of data from human cell-based MPS. We developed rat and dog quad-culture liver MPS incorporating primary hepatocytes, sinusoidal endothelial, Kupffer, and stellate cells. Using cryopreserved primary cells, we established a protocol for co-culturing cells under physiological flow conditions. Cells were evaluated for viability, morphology, and function (e.g. albumin production, cytochrome P450, and flavin-containing monooxygenase [FMO] activity). Optimized culture conditions maintained high-quality rat and dog liver chips for up to 7 days. Model performance was evaluated with ABT-288, a histamine-3 receptor antagonist that caused elevated serum transaminases in dogs but not rats. This finding was partially attributed to the high levels of FMO-mediated N-oxide metabolites produced in the dog. Key findings in our study were (i) dog chips showed much higher FMO-mediated N-oxidation compared with rat, and (2) dog chips exhibited modestly higher sensitivity to ABT-288 toxicity endpoints (albumin, alanine transaminase, and lactate dehydrogenase) compared with rat. Species differences in N-oxidation were not observed in rat and dog liver microsomes or 2D hepatocyte monocultures, suggesting that properties of the quad-culture MPS were necessary to model higher FMO activity observed in dogs in vivo. The data suggest that this preclinical species liver chip model provides novel understanding of in vitro to in vivo translation of ABT-288 dog liver toxicity.

While cytochrome P450 enzymes and UDP-glucuronosyltransferases play predominant roles in drug metabolism, hydrolases are emerging as key players in the metabolism of both small molecules and antibody-drug conjugates or peptide-oligonucleotide conjugates. Despite their importance, the protein levels of hydrolases across tissues and their inter-individual variability remain poorly understood. Although targeted proteomics can provide high selectivity and precision in quantifying small numbers of proteins, total protein approach (TPA)-based proteomics is emerging as a superior approach for multiplexed quantification of proteins. We performed a head-to-head comparison of targeted proteomics and TPA-based proteomics for quantifying 12 clinically relevant hydrolases in human liver and intestinal S9 fractions (N = 5 each). TPA-based global proteomics offered higher precision (coefficient of variation <20%), comparable sensitivity, along with its inherent advantage of a greater protein coverage than targeted proteomics. TPA data revealed the following order of protein abundance for target proteins: CES1> EPHX1 > CES2 > BPHL > PON3 > PON1 ∼ AADAC > CTSA > DPP4 in the liver and CES2 > ADA > DPEP1 ∼ AADAC ∼ EPHX1 > ALPI > DPP4 > CTSA > BPHL ∼ CES1 in the intestine. This study highlights the utility of TPA-based global proteomics for characterizing differential tissue abundance of hydrolases and their inter-individual variability.

Most complex in vitro models (CIVM) and microphysiological systems (MPS) are composed of human cells, with the goal of evaluating diseases, efficacy, safety, and pharmacokinetic ques­tions specifically for humans. The hope with CIVM/MPS is that they will eventually improve our predictivity of clinical responses and reduce or replace animal use in research, supporting the 3Rs concept of only using animals in research when necessary. Given the potential of animal-based models to advance this field by comparing existing in vivo animal data with new animal-based MPS responses, there are currently few CIVM and MPS utilizing animal tissues. Animal-based MPS may also have specific utility for cross-species comparisons or species-specific mechanistic questions on zoonotic diseases, and therapies for animals. Animal-based MPS may help expand in-vitro-to-in-vivo correlations, advance the field, and establish confidence in the predictive nature of such platforms. The IQ MPS-FDA workshop provided an interactive venue for pharmaceutical companies and regulatory agencies such as the U.S. Food and Drug Administration (FDA), NC3Rs (UK), Health Canada, NIH/NCATS, NIHS and PMDA (Japan), Danish Medicines Agency, European Com­mission, NIEHS/NICEATM, HHS, NIST, EURL ECVAM, and the IQ MPS Affiliate, a collaboration of pharmaceutical companies, to jointly discuss considerations of animal-based MPS and applica­tions where animal-based MPS are of potential value.

Hepatic clearance (CLH ) prediction is a critical parameter to estimate human dose. However, CLH underpredictions are common, especially for slowly metabolized drugs, and may be attributable to drug properties that pose challenges for conventional in vitro absorption, distribution, metabolism, and elimination (ADME) assays, resulting in nonvalid data, which prevents in vitro to in vivo extrapolation and CLH predictions. Other processes, including hepatocyte and biliary distribution via transporters, can also play significant roles in CLH Recent advances in understanding the interplay of metabolism and drug transport for clearance processes have aided in developing the extended clearance model. In this study, we demonstrate proof of concept of a novel two-step assay enabling the measurement of multiple kinetic parameters from a single experiment in plated human primary hepatocytes with and without transporter and cytochrome P450 inhibitors-the hepatocyte uptake and loss assay (HUpLA). HUpLA accurately predicted the CLH of eight of the nine drugs (within twofold of the observed CLH ). Distribution clearances were within threefold of observed literature values in standard uptake and efflux assays. In comparison, the conventional suspension hepatocyte stability assay poorly predicted the CLH The CLH of only two drugs was predicted within twofold of the observed CLH Therefore, HUpLA is advantageous by enabling the measurement of enzymatic and transport processes concurrently within the same system, alleviating the need for applying scaling factors independently. The use of primary human hepatocytes enables physiologically relevant exploration of transporter-enzyme interplay. Most importantly, HUpLA shows promise as a sensitive measure for low-turnover drugs. Further evaluation across different drug characteristics is needed to demonstrate method robustness. SIGNIFICANCE STATEMENT: The hepatocyte uptake and loss assay involves measuring four commonly derived in vitro hepatic clearance endpoints. Since endpoints are generated within a single test system, it blunts experimental error originating from assays otherwise conducted independently. A key advantage is the concept of removing drug-containing media following intracellular drug loading, enabling the measurement of drug reappearance rate in media as well as the measurement of loss of total drug in the test system unencumbered by background quantities of drug in media otherwise present in a conventional assay.

In vitro clearance assays are routinely conducted in drug discovery to predict in vivo clearance, but low metabolic turnover compounds are often difficult to evaluate. Hepatocyte spheroids can be cultured for days, achieving higher drug turnover, but have been hindered by limitations on cell number per well. Corning Elplasia microcavity 96-well microplates enable the culture of 79 hepatocyte spheroids per well. In this study, microcavity spheroid properties (size, hepatocyte function, longevity, culturing techniques) were assessed and optimized for clearance assays, which were then compared with microsomes, hepatocyte suspensions, two-dimensional-plated hepatocytes, and macrowell spheroids cultured as one per well. Higher enzyme activity coupled with greater hepatocyte concentrations in microcavity spheroids enabled measurable turnover of all 17 test compounds, unlike the other models that exhibited less drug turnover. Microcavity spheroids also predicted intrinsic clearance (CLint) and blood clearance (CLb) within threefold for 53% [9/17; average absolute fold error (AAFE), 3.9] and 82% (14/17; AAFE, 2.6) of compounds using a linear regression correction model, respectively. An alternate method incorporating mechanistic modeling that accounts for mass transport (permeability and diffusion) within spheroids demonstrated improved predictivity for CLint (12/17; AAFE, 4.0) and CLb (14/17; AAFE, 2.1) without the need for empirical scaling factors. The estimated fraction of drug metabolized by cytochrome P450 3A4 (fm,CYP3A4) using 3 μM itraconazole was within 25% of observed values for 6 of 8 compounds, with 5 of 8 compounds within 10%. In sum, spheroid cultures in microcavity plates permit the ability to test and predict clearance as well as fm,CYP3A4 of low metabolic turnover compounds and represent a valuable complement to conventional in vitro clearance assays. SIGNIFICANCE STATEMENT: Culturing multiple spheroids in ultralow attachment microcavities permits accurate quantitation of metabolically stable compounds in substrate depletion assays, overcoming limitations with singly cultured spheroids. In turn, this permits robust estimates of intrinsic clearance, which is improved with the consideration of mass transport within the spheroid. Incubations with 3 μM itraconazole enabled assessments of CYP3A4 involvement in hepatic clearance.

Incubation of drugs with suspension hepatocytes (SH) to determine intrinsic clearance is common in drug discovery. However, the limited duration of SH assays hampers clearance assessment of metabolically stable compounds. In turn, this has driven the development of alternative in vitro approaches to generate intrinsic clearance estimates. Culturing primary hepatocytes with supportive cells as co/tricultures has been shown to maintain morphology, viability, and drug-metabolizing enzyme function for weeks, permitting extended incubations. Another assay from our laboratory is the preloaded hepatocyte assay (preload assay), which involves preloading plated monoculture hepatocytes with compounds and measuring the loss from cells in drug-free media. This approach increases analytical sensitivity compared to assays that measure bulk compound loss in the cells plus medium. We conducted a systematic evaluation of the ability of coculture, triculture, and preload assay models to predict human in vivo clearance for 50 predominantly low-clearance compounds with a range of physicochemical properties, including equal numbers of compounds following or violating Lipinski's rule of 5, across 3 hepatocyte donors. The results were compared with SH. Co/tricultures exhibited lower inter-donor differences compared to the preload and SH assays, likely due to the blunting of environmental cues after 5 days in culture prior to compound introduction. All 3 plated models significantly reduced the number of compounds with insufficient turnover to calculate CLint,u compared to SH (SH: 40%; preload: 18%; cocultures: 8%; tricultures: 4%), exhibited strong interexperimental reproducibility and robust predictions of blood clearance (preload: 26/41; cocultures: 31/46; tricultures: 30/48 within 3-fold of observed). SIGNIFICANCE STATEMENT: Preloading plated hepatocytes with compounds and measuring the loss in drug-free media, or culturing hepatocytes with supportive cells as co/tricultures, facilitate quantitation of metabolically stable compounds in substrate depletion assays compared to suspension hepatocytes (SH). All 4 models exhibit robust estimates of CLint,u and CLb, but plated models allowed assessment of several compounds found to be too stable to evaluate in SH.

Permeability is a key factor driving the absorption of orally administered drugs. In early discovery, the efficient evaluation of permeability, particularly for compounds violating Lipinski's Rule of 5, remains challenging. Addressing this, we established a high-throughput method to measure the experimental polar surface area (HT-EPSA) as an in vitro surrogate to measure permeability. Compared to earlier methods, HT-EPSA significantly reduces data acquisition time with enhanced sensitivity, selectivity, and data quality. In the effort of translating EPSA to human in vitro and in vivo passive permeability, we demonstrated the application of EPSA for predicting Caco-2 cell and human intestinal permeability, showing improvements over topological polar surface area and the parallel artificial membrane permeability assay for rank-ordering permeability in a proteolysis targeting chimera case study. The HT-EPSA method is expected to be highly beneficial in guiding early stage compound rank-ordering, faster decision-making, and in predicting in vitro and/or in vivo human intestinal permeability.

Pharmaceutical companies continue to advocate for the use of in vitro models towards the reduction of animal use in drug discovery and development while acknowledging that further advancements are needed to heighten the models’ current state of readiness.

Microphysiological systems (MPS) incorporating human intestinal organoids have shown the potential to faithfully model intestinal biology with the promise to accelerate development of oral prodrugs. We hypothesized that an MPS model incorporating flow, shear stress, and vasculature could provide more reliable measures of prodrug bioconversion and permeability. Following construction of jejunal and duodenal organoid MPS derived from 3 donors, we determined the area under the concentration-time (AUC) curve for the active drug in the vascular channel and characterized the enzymology of prodrug bioconversion. Fosamprenavir underwent phosphatase mediated hydrolysis to amprenavir while dabigatran etexilate (DABE) exhibited proper CES2- and, as anticipated, not CES1-mediated de-esterification, followed by permeation of amprenavir to the vascular channel. When experiments were conducted in the presence of bio-converting enzyme inhibitors (orthovanadate for alkaline phosphatase; bis(p-nitrophenyl)phosphate for carboxylesterase), the AUC of the active drug decreased accordingly in the vascular channel. In addition to functional analysis, the MPS was characterized through imaging and proteomic analysis. Imaging revealed proper expression and localization of epithelial, endothelial, tight junction and catalytic enzyme markers. Global proteomic analysis was used to analyze the MPS model and 3 comparator sources: an organoid-based transwell model (which was also evaluated for function), Matrigel embedded organoids and finally jejunal and duodenal cadaver tissues collected from 3 donors. Hierarchical clustering analysis (HCA) and principal component analysis (PCA) of global proteomic data demonstrated that all organoid-based models exhibited strong similarity and were distinct from tissues. Intestinal organoids in the MPS model exhibited strong similarity to human tissue for key epithelial markers via HCA. Quantitative proteomic analysis showed higher expression of key prodrug converting and drug metabolizing enzymes in MPS-derived organoids compared to tissues, organoids in Matrigel, and organoids on transwells. When comparing organoids from MPS and transwells, expression of intestinal alkaline phosphatase (ALPI), carboxylesterase (CES)2, cytochrome P450 3A4 (CYP3A4) and sucrase isomaltase (SI) was 2.97-, 1.2-, 11.3-, and 27.7-fold higher for duodenum and 7.7-, 4.6-, 18.1-, and 112.2-fold higher for jejunum organoids in MPS, respectively. The MPS approach can provide a more physiological system than enzymes, organoids, and organoids on transwells for pharmacokinetic analysis of prodrugs that account for 10% of all commercial medicines. We sought to develop a microphysiological intestinal test system for predicting oral prodrug bioconversion and permeability in humans. The investigation included extensive comparisons of candidate in vitro cell models and deep proteomic analysis.

According to the free drug hypothesis (FDH), only free, unbound drug is available to interact with biological targets. This hypothesis is the fundamental principle that continues to explain the vast majority of all pharmacokinetic and pharmacodynamic processes. Under the FDH, the free drug concentration at the target site is considered the driver of pharmacodynamic activity and pharmacokinetic processes. However, deviations from the FDH are observed in hepatic uptake and clearance predictions, where observed unbound intrinsic hepatic clearance (CLint,u) is larger than expected. Such deviations are commonly observed when plasma proteins are present and form the basis of the so-called plasma protein-mediated uptake effect (PMUE). This review will discuss the basis of plasma protein binding as it pertains to hepatic clearance based on the FDH, as well as several hypotheses that may explain the underlying mechanisms of PMUE. Notably, some, but not all, potential mechanisms remained aligned with the FDH. Finally, we will outline possible experimental strategies to elucidate PMUE mechanisms. Understanding the mechanisms of PMUE and its potential contribution to clearance underprediction is vital to improving the drug development process.

Objective: This work describes a simplified, 96-well plate method for determining the blood-to-plasma concentration ratio (BP ratio) for small molecules. Methods: The need for calibration curves was eliminated using a matrix-matching approach in which blood samples were mixed with blank plasma and plasma samples were mixed with blank blood. As a result, both blood- and plasma-origin samples shared an equivalent matrix ahead of bioanalysis. In the in vitro assay, identical sample matrices were achieved by using the same source of blank plasma and blood. Results: In humans, a good correlation (R2 = 0.84) was observed between the data obtained in this ma-trix-matching method and literature values for 11 commercial compounds possessing a wide range of logD values across multiple chemical classes. In addition, this method showed good agreement with in vitro BP ratios for 10 proprietary compounds determined radiometrically (R2 = 0.72) in human and preclinical species. Finally, the in vitro matrix matching method compared favorably to BP ratios de-termined ex vivo for 13 proprietary and literature compounds (R2 = 0.87) in rat. Conclusion: This method, suitable for in vitro and ex vivo BP ratio determinations, is operationally efficient, robust, and a useful improvement upon previously published methods.

Complex in vitro models (CIVM) offer the potential to improve pharmaceutical clinical drug attrition due to safety and/ or efficacy concerns. For this technology to have an impact, the establishment of robust characterization and qualifi­cation plans constructed around specific contexts of use (COU) is required. This article covers the output from a workshop between the Food and Drug Administration (FDA) and Innovation and Quality Microphysiological Systems (IQ MPS) Affiliate. The intent of the workshop was to understand how CIVM technologies are currently being applied by pharma­ceutical companies during drug development and are being tested at the FDA through various case studies in order to identify hurdles (real or perceived) to the adoption of microphysiological systems (MPS) technologies, and to address evaluation/qualification pathways for these technologies. Output from the workshop includes the alignment on a working definition of MPS, a detailed description of the eleven CIVM case studies presented at the workshop, in-depth analysis, and key take aways from breakout sessions on ADME (absorption, distribution, metabolism, and excretion), pharmacology, and safety that covered topics such as qualification and performance criteria, species differences and concordance, and how industry can overcome barriers to regulatory submission of CIVM data. In conclusion, IQ MPS Affiliate and FDA scientists were able to build a general consensus on the need for animal CIVMs for preclinical species to better determine species concordance. Furthermore, there was acceptance that CIVM technologies for use in ADME, pharmacology and safety assessment will require qualification, which will vary depending on the specific COU.

Screening for cytochrome P450 (CYP) induction potential is routine in drug development. Induction results in a net increase in CYP protein and is assessed typically by measuring indirect endpoints, i.e., enzyme activity and mRNA in vitro. Recent methodological advancements have made CYP protein quantification by liquid chromatography-mass spectrometry in vitro induction studies more accessible and amenable to routine testing. In this study, we evaluated CYP3A4 concentration dependence of induction response for 11 compounds (rifampin, rifabutin, carbamazepine, efavirenz, nitrendipine, flumazenil, pioglitazone, rosiglitazone, troglitazone, pazopanib, and ticagrelor) in plated hepatocytes from two or three donors incorporating in the assessment all three endpoints. In addition, the time-dependence of the induction was examined over 1, 2, or 3 days of treatment. For most compounds, mRNA, enzyme activity, and protein endpoints exhibited similarity in induction responses. Pazopanib and ticagrelor were notable exceptions as neither protein nor enzyme activity were induced despite mRNA induction of a magnitude similar to efavirenz, pioglitazone, or rosiglitazone, which clearly induced in all three endpoints. Static modeling of clinical induction responses supported a role for protein as a predictive endpoint. These data highlight the value of including CYP protein quantification as an induction assay endpoint to provide a more comprehensive assessment of induction liability. SIGNIFICANCE STATEMENT: Direct, liquid chromatography-mass spectrometry (LC-MS)-based quantification of cytochrome P450 (CYP) protein is a desirable induction assay endpoint; however such application has been limited due to inefficient workflows. Here, we incorporate recent advancements in protein quantitation methods to efficiently quantify CYP3A4 protein in in vitro induction assays with 11 compounds in up to 3 donors. The data indicate induction responses from mRNA do not always align with those of protein suggesting assessment of induction liability is more complex than thought previously.

In this chapter, we illustrate the criticality of proper fitting of enzyme kinetic data. Simple techniques are provided to arrive at meaningful kinetic parameters, illustrated using an example, nonmonotonic data set. In the initial analysis of this data set, derived Km and Vmax parameters incorporated into PBPK models resulted in outcomes that did not adequately describe clinical data. This prompted a re-review of the in vitro data set and curve-fitting procedures. During this review, it was found that the 3-parameter model was fitted on data that was improperly unweighted. Reanalysis of the data using a weighted model returned a better fit and resulted in kinetic parameters better aligning with clinical data. Tools and techniques used to identify and compare kinetic models of this data set are provided, including various replots, visual inspection, examination of residuals, and the Akaike information criterion.

A method for obtaining spectral quantitation of cytochrome P450 in microsomes using a single-beam spectrophotometer is described. The method involves obtaining the difference spectrum of the reduced form of cytochrome P450 subtracted from the spectrum of the carbon monoxide bound, reduced form. The resulting spectrum can then be analyzed to determine the difference between the absorbance at 450 nm and an isosbestic value (usually at 490 nm). The difference is then divided by the extinction coefficient of 91 mM(-1) cm(-1) to obtain nmol P450 per mL. The method described entails the use of a single-beam instrument where the total P450 enzyme is reduced by sodium dithionite prior to equilibration of the sample with carbon monoxide. Variations on the method to reduce the potentially confounding effects of hemoglobin contamination are also included.