Activity of CYP2E1 and CYP3A enzymes in adults with moderate alcohol consumption: A comparison with nonalcoholics

Authors


  • Potential conflict of interest: Nothing to report.

Abstract

Alcohol consumption is known to induce hepatic CYP2E1 activity, but its effect on hepatic and intestinal CYP3A in humans is not known. We have conducted a study to compare the CYP2E1 and CYP3A activities in 20 individuals with moderate alcohol consumption and 20 gender-, race-. and body mass index (BMI)-matched nonalcoholics. Intravenous and oral midazolam (MDZ) clearances were used to measure the in vivo CYP3A activity, and chlorzoxazone (CHZ) oral clearance was used to assess in vivo CYP2E1 activity. Furthermore, we assessed the relationship between hepatic CYP2E1 and CYP3A activities and their messenger RNA (mRNA) expression in the peripheral lymphocytes. The systemic clearance (CL) of MDZ was not different between alcoholics (36.9 ± 12 L/hr) and nonalcoholics (36.6 ± 14.1; P = .9). The oral availability of MDZ was significantly lower in alcoholics than in the nonalcoholics (0.28 ± .09 vs. 0.38 ± .17, respectively, P = .03). The maximum serum concentration after oral midazolam dosing was significantly different between the 2 groups. CHZ CL was significantly higher in alcoholics than in nonalcoholics (31.5 ± 11.9 vs. 23.4 ± 8.7 L/hr, P < 0.05). CYP3A4 and CYP2E1 mRNA levels were not significantly different between the groups, and no correlation was observed between lymphocyte CYP mRNA and in vivo CYP activity. In conclusion, in individuals with moderate alcohol consumption, there was no alteration in the hepatic CYP3A activity, but the reduced midazolam oral bioavailability suggests that moderate alcohol consumption may cause intestinal CYP3A induction. Lymphocyte CYP2E1 and CYP3A4 mRNA levels did not correlate with CYP2E1 and CYP3A activities. (HEPATOLOGY 2005;41:1144–1150.)

Hepatic CYP2E1 can be induced up to eight-fold by ethanol in rodents, and its activity is elevated in association with consumption of alcoholic beverages in humans. The increased CYP2E1 activity may play a role in the pathogenesis of alcoholic liver disease as well as in the pathogenesis of alcohol-mediated increase in the risk of acetaminophen hepatotoxicity.1–3 However, ethanol and other alcohols (e.g., isopentanol) have also been shown to induce CYP3A in several experimental systems, including primary cultures of rat hepatocytes, a rat hepatoma cell line, and intact rats.4–7 This is important because CYP3A has been shown to be an important contributor to the formation of the reactive acetaminophen metabolite, N-acetyl-p-benzoquinone imine (NAPQI), believed to cause hepatotoxicity.8 The ability of alcohol to induce CYP3A in humans is less well-defined. Hoshino and Kawasaki demonstrated that alcoholics, admitted to the hospital for detoxification, had an increased 6β-hydroxycortisol to 17-hydroxycortisol ratio compared with normal volunteers, which declined significantly after detoxification of the patients.9, 10 Likewise, alcoholics require greater amounts of the CYP3A substrate, fentanyl, to achieve adequate analgesia compared with nonalcoholics.11 The need for dose supplementation for CYP3A substrates in alcoholics appears to be necessary for other agents, such as midazolam (MDZ), used for conscious sedation before endoscopy.

Although little is known about racial differences in CYP2E1 activity between African Americans and Caucasians, some evidence suggests racial differences in CYP3A activity. For example, the bioavailability of cyclosporine a CYP3A and MDR1 substrate is significantly reduced in African Americans compared with Caucasians.12–15 Other immunosuppressants, such as tacrolimus, sirolimus, which are also CYP3A and MDR1 substrates, are similarly affected after administration to African Americans.16–20 Although the mechanism of the reduced immunosuppressant bioavailability is not well-established, it is thought that it is related to an individual's MDR1 genotype.21 Wandel et al.22 reported that the systemic clearance of MDZ, a prototypic CYP3A substrate, was reduced, but not the oral clearance of MDZ, in African Americans compared with European Americans. However, others have reported that the systemic and oral clearance of MDZ were not significantly different between African Americans and European Americans.23

Therefore, we have investigated the role of moderate alcohol consumption (140 g ethanol per week) on CYP3A and CYP2E1 messenger RNA (mRNA) levels and activity in vivo using the CYP3A probe MDZ and CYP2E1 probe chlorzoxazone (CHZ) in 20 alcoholics and 20 nonalcoholics. Additionally, many of the hepatic phase I and phase II enzymes are expressed in the lymphocytes. Lymphocyte mRNA levels may serve as surrogate for activity of drug metabolizing enzymes, but the utility of this approach is questionable. We therefore evaluated whether CYP2E1 and CYP3A4 mRNA expression in lymphocytes would reflect the in vivo activity of these enzymes.

Abbreviations

MDZ, midazolam; CHZ, chlorzoxazone;: mRNA, messenger RNA; PCR, polymerase chain reaction.

Patients and Methods

This study was reviewed and approved by the Clarian Health Partners and Indiana University Purdue University Indianapolis Institutional Review Board and the Advisory Committee for the General Clinical Research Center of Indiana University School of Medicine. All participants provided written informed consent.

Participants.

Twenty individuals with moderate alcohol consumption (alcohol group) and 20 age- and gender-matched individuals without alcohol consumption (nonalcohol group) participated in this study. The reported alcohol consumption in participants belonging to the alcohol group ranged from140 to 210 g per week (average, 2–3 drinks per day), and all of them have regularly consumed alcohol for more than 5 years. In individuals who reported alcohol consumption, the quantity of their consumption was confirmed with a family member or a friend. In individuals who reported no alcohol consumption, the absence of alcohol consumption was confirmed by CAGE questionnaire and with a family member or a friend. Volunteers were determined to be free of other significant medical conditions as assessed by medical history, physical examination including electrocardiogram, and blood and urine chemistry screens. Participant demographics are presented in Table 1.

Table 1. Demographics and Selected Clinical Characteristics of Study Participants*
 Alcohol Group (n = 20)Nonalcohol Group (n = 20)
  • Abbreviations: BMI, body mass index; MCV, mean corpuscular volume; AST, aspartate aminotransferase.

  • *

    Values are expressed as mean ± SD unless otherwise specified.

  • MCV was significantly higher in alcohol group than the control group (P = .006).

Age (yrs)31.6 ± 7.332.1 ± 8.7
Sex (M/F)16/416/4
Race  
 African American1010
 Caucasian1010
Weight (kg)91 ± 2595 ± 24
Height (cm)178 ± 9.4177 ± 9.8
BMI (kg/m2)28.5 ± 630 ± 6.4
White cell count (×103 per mm3)6.9 ± 1.76.3 ± 1.7
Hemoglobin (g/dL)14.6 ± 1.014.5 ± 1.2
MCV (fL)92 ± 3.686 ± 8
Platelet count (×103 per mm3)262 ± 54.5266 ± 56
AST (U/L)27 ± 728 ± 8
Alkaline phosphatase (U/L)74 ± 2169 ± 13
Bilirubin (mg/dL)0.3 ± 0.20.6 ± 0.4
Albumin (g/dL)3.9 ± 0.23.9 ± 0.3
Creatinine (mg/dL)0.9 ± 0.11.0 ± 0.2

Study Design.

Alcohol consumption before participation in the study was not controlled. For at least 48 hours before the study, participants were instructed to avoid consuming certain foods such as grapefruit (and other citrus fruits), charbroiled meats, cruciferous vegetables (e.g., kale, broccoli, collard greens, mustard greens, etc.), beverages such as grapefruit juice, tea, coffee, colas (i.e., Coke, Mountain Dew), and over-the-counter medications including herbal remedies (e.g., acetaminophen, St. John's wort) as well as prescription medications. After an overnight fast, participants emptied their bladder, and a baseline blood sample was obtained. Then, participants received MDZ (0.05 mg/kg over 30 minutes) intravenously and 5 mg orally on consecutive days. Serial blood samples were obtained from the contralateral arm 15, 30, 45, 60 minutes and 1½, 2, 2½, 3, 4, 6, and 8 hours after the start of MDZ dosing. Individuals were given standard meal (without the food previously described) after 4 hours of MDZ dosing. CHZ (500 mg) was administered orally 8 hours after the IV dose of MDZ on day 1. Serial blood samples were obtained at 15, 30, and 60 minutes, and 1½, 2, 3, 4, 6, and 8 hours after CHZ dosing. Participants were discharged after collection of the final blood sample. All of the participants reported compliance with the recommended dietary and medicinal restrictions. Samples were stored at −20°C until analysis.

Quantitation of Midazolam and Its Metabolites.

MDZ and its metabolites were quantified in serum by liquid chromatography-mass spectrometry using desmethyldiazepam as the internal standard as previously described.24 The interday coefficient of variation at 1.4, 18, and 50 ng/mL was 9.2%, 6.7%, and 8.9%, respectively. The relative error for quality control samples with nominal values of 1.4, 18, and 50 ng/mL was −1.0%, 3.3%, and −2.7%, respectively.

Determination of Chlorzoxazone Serum Concentrations.

The serum concentrations of CHZ were determined using high-performance liquid chromatography with ultraviolet dectection at 274 nm as previously described.25, 26 The interday coefficient of variation at 0.14, 1.4, and 8 μg/mL was 6.9%, 6.2%, and 7.6%, respectively. The relative error for quality control samples with nominal values of 0.14, 1.4, and 8 μg/mL was 1.4%, −4.5%, and −1.3%, respectively. Likewise, urinary 6-hydroxychlorzoxazone concentrations were determined after adding 2,000 units β-glucuronidase to 0.5 mL urine and incubating for 1 hour at 37°C. Samples were processed as described previously by Wang et al.25

Peripheral Lymphocyte mRNA Quantitation.

Lymphocytes were isolated from 25 mL heparinized blood, using 10 mL Isolymph (Gallard-Schlesinger Industries, Carle Place, NY) per the manufacturer's instruction. Mononuclear cells containing mainly lymphocytes (>75%) and 12% to 25% monocytes were counted, and the remaining portion of pellet was dissolved in TRI Reagent and stored at −80°C until RNA isolation.

Total RNA from lymphocytes was isolated using Trizol® reagent (Invitrogen Corp., Carlsbad, CA) according the manufacturer's instructions. RNA was quantified by measuring the absorbance at 260 nm (Beckman DU 640, Beckman Coulter, Inc., Fullerton, CA) and quality assessed by calculated 260 nm/280 nm ratio. After RNA isolation, all samples were stored at −80°C until complementary DNA (cDNA) preparation. In a 20-μL reaction containing random hexamers, 2 μg total RNA was reverse transcribed into cDNA, using the Promega Reverse Transcription system (Promega Corp., Madison, WI) according to the manufacturer's instructions.

CYP3A4 and GAPDH.

A real-time polymerase chain reaction (PCR) method was used to measure the amount of CYP3A4 mRNA as previously described with minor modifications.27 Primers specific to CYP3A4 and GAPDH transcripts were purchased from Integrated DNA Technologies (Coralville, IA). The CYP3A4 forward primer sequence was 5′- CAT TCC TCA TCC CAA TTC TTG AAG T –3′, and the reverse primer sequence was 5′- CCA CTC GGT GCT TTT GTG TAT CT –3′. Each sample was normalized to expression of the housekeeping gene GAPDH, using the forward primer 5′- GAA GGT GAA GGT CGG AGT C –3′ and the reverse primer 5′- GAA GAT GGT GAT GGG ATT TC –3′. The PCR mixture contained UDG Platinum supermix reagent and forward and reverse primer (200 nmol/L each). Amplification and detection was performed using an Icycler (Bio-Rad, Hercules, CA) under the following conditions: 50°C for 2 minutes and then 42 cycles of 95°C for 15 seconds and 60°C for 1 minute. Detection of CYP3A4 and GAPDH was done with SYBR green Core Reagents (Applied Biosytems, Foster City, CA). Calibration curves were generated by serial dilutions of CYP3A4 and GAPDH (200 fg–0.2 ag), and mRNA expression of CYP3A4 was normalized to GAPDH mRNA expression for each participant.

CYP2E1.

A real-time PCR method was used to measure the amount of CYP2E1 mRNA as previously described with minor modifications.28 Primers specific to CYP2E1 transcripts were purchased from Integrated DNA Technologies. The CYP2E1 forward primer sequence was 5′-AAC TGT CCC CGG GAC CTC-3′ reverse primer 5′-GCG CTC TGC ACT GTG CTT T-3′ and fluorescent probe 5′ FAM-CCA TTT CCA CGA GCA GGC AGT CG-BHQ 3′. The PCR mixture contained Taqman Universal Master Mix reagent, forward and reverse primer (300 nmol/L each) and 200 nmol/L fluorescent probe. Amplification and detection was performed using an Icycler (Bio-Rad) under the following conditions: 50°C for 2 minutes, 95°C for 10 minutes, and then 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. Calibration curves were generated by serial dilutions of CYP2E1 and GAPDH (200 fg-0.2 ag), and mRNA expression of CYP2E1 was normalized to GAPDH mRNA expression for each participant.

Pharmacokinetic and Statistical Analysis.

Data are reported as mean ± SD. Blood concentrations of MDZ were determined as previously described.24 The pharmacokinetics of MDZ and CHZ were determined by using noncompartmental methods (Winnonlin v. 4.0, Pharsight Corp, Mountain View, CA). The oral availability of MDZ was calculated by using equation 1:

equation image(1)

With the assumption that liver is the sole organ of elimination after systemic administration of drug, the hepatic availability of MDZ after intravenous administration was calculated using equation 2:

equation image(2)

Equation 3 was used to calculate the intestinal availability, which assumes that the fraction of dose absorbed into the intestinal wall is equal to 1:

equation image(3)

The effect of alcohol on the pharmacokinetics of MDZ and CHZ (e.g., clearance, availability) was determined with the use of descriptive statistics and Student t test as appropriate, using the JMP computer program (version 4.02, SAS Institute, Cary, NC). Spearman rank correlations were used to detect the associations between hepatic CYP2E1 and CYP3A activity and their peripheral lymphocyte mRNA expression. The primary end point for this study was the systemic clearance of MDZ, and secondary end points were oral clearance of MDZ and CHZ. A sample size of 20 individuals in each group will require a 30% difference in systemic clearance between the groups to have a power of 80% at the .05 significance level, and, based on our studies, this difference was thought to be biologically plausible.24 A P value less than .05 was considered statistically significant.

Results

Forty participants successfully completed the study without reporting any significant adverse effects from the CHZ and MDZ dosing. Participants in the alcohol group reported alcohol consumption in the evening before their admission to the General Clinical Research Center. The disposition of CHZ after oral dosing is shown in Fig. 1A. Figure 2 shows the disposition of MDZ after intravenous and oral administration in the alcohol and control groups. Moderate alcohol consumption significantly increased the oral clearance of CHZ (Table 2 and Fig. 1B) and reduced the area under the concentration time curve (Table 2). The elimination half-life for CHZ was unaffected by moderate alcohol consumption. The urinary recovery of 6-hydroxychlorzoxazone as a percent of the dose administered was not significantly different between alcohol and control groups (65 ± 24% vs. 61 ± 22%, P = .57).

Figure 1.

(A) The disposition of chlorzoxazone after oral administration in 20 individuals in the alcohol group and 20 nonalcoholic individuals and (B) the effect of alcohol consumption on the oral clearance of chlorzoxazone. Open squares, Nonalcoholics; solid circles, Alcoholics. Points and bars represent mean (±SD). *Significant (P = .018) difference in the oral chlorzoxazone clearance.

Figure 2.

The disposition of midazolam after (A) intravenous and (B) oral administration in 20 individuals in the alcohol group and 20 individuals with no alcohol consumption. Open squares, Nonalcoholics; and solid circles, Alcoholics. Points represent the mean (±SD) of 17 to 20 observations.

Table 2. Pharmacokinetic Parameters (Mean ± SD) for Chlorzoxazone (In Vivo Probe for Hepatic CYP2E1 Activity) After Its Oral Administration and Peripheral Lymphocyte CYP2E1 mRNA Expression
 Alcoholics (n = 20)Nonalcoholics (n = 20)
  • Abbreviations: AUC, area under the curve; CLpo, oral clearance; T ½, half-life; Cmax, maximum serum concentration; Tmax, time to reach maximum serum concentration.

  • *

    Significant difference between alcohol and the nonalcohol groups (P ≤ .05).

  • Median and range

  • CYP2E1 mRNA was normalized against GAPDH mRNA; measured in 18 alcoholics and 17 nonalcoholics.

AUCmath image (hr × mg/L)17.7 ± 5.3*24.5 ± 9.6
CLPO (L/hr)31.5 ± 11.9*23.4 ± 8.7
(L/hr/kg)0.37 ± 0.17*0.26 ± 0.12
T ½ (hrs)1.4 ± 0.71.8 ± 0.70
Cmax (mg/L)7.1 ± 3.38.0 ± 3.8
Tmax (hr)1.75 (0.5, 4.0)1.75 (1.0, 4.0)
Lymphocyte CYP2E1 mRNA29.7 × 10−5 ± 44.19 × 10−516.36 × 10−5 ± 17.8 × 10−5

Moderate alcohol consumption did not alter the disposition of MDZ after intravenous administration (Table 3; Fig. 2). The maximum serum concentration after oral MDZ dosing was significantly different between the 2 groups. The systemic and oral clearance of MDZ was not altered by moderate alcohol intake (Fig. 3A; Table 3). However, the oral availability of MDZ (FPO) was significantly (P = .031) reduced in the alcohol group (0.28 ± .09) compared with the controls (0.38 ± 0.17; Table 3 and Fig. 3B). Despite the reduction in oral availability, hepatic (P = .74) and intestinal (P = .07) availabilities were not significantly different between the 2 groups (Fig. 3B; Table 3).

Table 3. Pharmacokinetic Parameters (Mean ± SD) of Oral and Intravenous Midazolam: Assessment of Hepatic and Intestinal CYP3A Activity, and Peripheral Lymphocyte CYP3A4 mRNA Expression
 Alcoholics (n = 20)Nonalcoholics (n = 20)
  • Abbreviations: CLIV, systemic clearance; CLPO, Oral clearance; T ½, half-life; Cmax, maximum serum concentration; FPO, oral bioavailability; FH, hepatic availability; FG, intestinal availability.

  • *

    Significant difference between alcohol and nonalcohol groups (P ≤ .05).

  • CYP3A4 mRNA was normalized against GAPDH mRNA; measured in 15 alcoholics and 12 nonalcoholics.

CLIV (L/hr)36.9 ± 12.036.6 ± 14.1
T ½ (hrs)5.1 ± 2.86.2 ± 3.6
CLPO (L/hr)149 ± 88120.0 ± 84.0
 Cmax15.5 ± 5.5*20.7 ± 8.7
 FPO0.28 ± 0.09*0.38 ± 0.17
 FH0.72 ± 0.120.73 ± 0.13
 FG0.40 ± 0.140.54 ± 0.29
Lymphocyte CYP3A4 mRNA12 × 10−5 ± 20.4 × 10−54.15 × 10−5 ± 2.53 × 10−5
Figure 3.

The effect of moderate alcohol consumption (≥140 g ethanol/week) on the systemic (solid bars) and oral clearance (hatched bars) of (A) midazolam and (B) oral, hepatic, and intestinal availability of midazolam. Each bar represents the mean (±SD) of 20 values. *Significant (P = .031) difference between alcoholics and nonalcoholics.

The peripheral lymphocyte mRNA levels of CYP2E1 and CYP3A4 in alcohol and the control groups are shown in Tables 2 and 3, respectively, and Fig. 4. The peripheral lymphocyte CYP2E1 or CYP3A4mRNA expression was not significantly different between the alcohol and control groups (Table 2 and 3). No correlation was observed between peripheral lymphocyte CYP3A4 mRNA expression and CYP3A activity (r = −0.126) or between peripheral lymphocyte CYP2E1 mRNA expression and the hepatic CYP2E1 activity (r = 0.07).

Figure 4.

Peripheral lymphocyte CYP2E1 and CYP3A4 mRNA expression in alcoholics and nonalcoholics. Data represent peripheral lymphocytic expression of CYP2E1 (hatched bars) and CYP3A4 (solid bars) normalized against GAPDH, used a housekeeping gene. Each bar represents the mean (±SD).

Discussion

This human study makes several observations. First, it confirms the previous observation that even moderate alcohol consumption leads to significantly higher hepatic CYP2E1 activity in humans. However, we did not find a significant correlation between hepatic CYP2E1 activity and its expression in the peripheral lymphocytes, and this is consistent with our prior observations in patients with diabetes and obese patients with nonalcoholic fatty liver disease.25, 26 Our observations in moderate alcoholics differ from those of Raucy et al.,29 who showed a strong correlation between hepatic CYP2E1 activity and its expression in the peripheral lymphocytes in patients with heavy alcohol consumption. The reasons for this discrepancy are not clear but could be due in part to the fact that we studied individuals with moderate alcohol consumption, whereas individuals in the study by Raucy et al.29 had heavy alcohol consumption.

Second, although participants with moderate alcohol consumption did not have higher hepatic CYP3A activity, they had significantly higher intestinal CYP3A activity than those without alcohol consumption. Thus, from equation 3, we can say that the oral availability reflects the intestinal availability, and a high correlation (r = 0.92) was observed between oral and intestinal availabilities, which raises the possibility that alcohol may induce CYP3A at the small bowel level. This finding becomes very important for drugs that depend highly on gut wall CYP3A4 for their metabolism. For such drugs, coadministration with chronic ethanol intake could lead to increased gut wall metabolism by CYP3A4. Although studies are lacking that specifically examined the effect of alcohol consumption on small bowel CYPs, alcohol consumption has been shown to induce certain small bowel enzymes.30, 31 Garcia-Puges et al.32 have shown that alcoholic patients had significantly higher levels of gamma glutamyltranspeptidase compared with nonalcoholics, and the serum concentration of gamma glutamyl transpeptidase decreased rapidly after abstinence. More research is needed to explore the effect of varying amounts of alcohol consumption on small bowel CYPs and its possible implications.

The literature suggests that alcohol consumption may modulate hepatic CYP3A activity. Using urinary 6β-OH-cortisol/cortisol ratio as marker, 2 previous studies have suggested that CYP3A activity is higher in individuals with alcohol consumption than in the nonalcoholic volunteers.9, 10 In another study, the amount of fentanyl (a CYP3A substrate) required to achieve satisfactory analgesia was 61% higher in heavy alcoholics than in the nonalcoholic patients, suggesting that resistance to fentanyl in alcoholics could be from alcohol-mediated increase in hepatic CYP3A activity.11 Similarly, our anecdotal experience has been that alcoholics require far higher doses of intravenous MDZ to achieve sedation before gastrointestinal endoscopy. Based on these data, we anticipated that even moderate alcohol consumption might have some effect on hepatic CYP3A activity; however, our study showed that moderate alcohol consumption does not affect the hepatic CYP3A activity. It would be of interest to investigate the effect of heavy alcohol consumption on hepatic CYP activity; however, in our experience, most of these individuals have evidence of alcoholic liver injury. This problem makes it difficult to study the effects of heavy alcohol consumption per se on the in vivo activity of hepatic CYPs. Wolbold et al33 have shown that females have higher levels of CYP3A4, and therefore we reanalyzed our results to examine whether gender had significant effects on our results. When the data were reanalyzed without female participants (four per group), our results related to pharmacokinetic parameters or the mRNA expression were unchanged (data not shown).

Third, we observed that no correlation existed between the hepatic activity of CYP3A and CYP2E1 and their expression in the peripheral lymphocytes. CYP2E1 is known to be stabilized by ethanol, which leads to its longer half-life in the presence of ethanol.34 Because there is protein stabilization of CYP2E1 in addition to upregulation at the transcriptional level, lack of correlation was observed between CYP2E1 activity and mRNA expression in peripheral lymphocytes. Like CYP2E1, CYP3A4 is also a leaky enzyme that in the absence of substrate generates reactive oxygen species that increase enzyme degradation. Ethanol causes the substrate stabilization of CYP2E1. Similarly, ethanol also could cause substrate coupling with CYP3A4 and preserve it through substrate stabilization. Whether ethanol is a substrate for CYP3A4 is not known, but it is a small molecule and could easily fit into the big active site of CYP3A4. Feierman et al.7 have demonstrated using fentanyl as a probe that ethanol induced CYP3A activity. They have also reported increased expression of CYP3A4 protein as well as mRNA in HepG2 cells as a result of stabilization of protein and of mRNA.

Our study design deserves further discussion. In this study, we measured the activity of CYP2E1 and CYP3A enzymes in moderate alcoholics and compared them with matched controls without alcohol consumption. The ideal study design to examine the effect of moderate alcohol consumption on CYP2E1 and CYP3A activities is by measuring their activity before and after exposing them to moderate amounts of alcohol. However, conducting such a study is difficult, as our institutional review board opined that chronic administration of 2 to 3 alcoholic drinks per day to nondrinking research volunteers in a nonhospitalized setting was not acceptable.

In conclusion, moderate alcohol consumption does not alter the systemic clearance of MDZ but reduces MDZ oral bioavailability consistent with intestinal CYP3A induction. Lymphocyte CYP2E1 and CYP3A4 mRNA are not good surrogate markers for predicting hepatic CYP2E1 and hepatic and intestinal CYP3A activities in vivo.

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