Materials and Methods

Volunteers.

Twenty-two subjects (10 men and 12 women, ranging in age from 20 to 50 years) participated in this investigation. Volunteers were healthy subjects within 20% of ideal body weight. Individuals with diabetes, individuals receiving prescription medications, or individuals diagnosed with a codependency were eliminated from the study. At the time of recruitment, informed consent was obtained, questionnaires were completed, and a blood sample was drawn for routine blood evaluations (Reference Laboratory, Inc., Albuquerque, NM). Based on laboratory results, subjects demonstrating abnormal liver function values or those diagnosed with hepatic disease were discharged. Volunteers proceeding with the investigation were admitted to the University of New Mexico Clinical Research Center, where they received thorough physical examinations. Subjects remained overnight in the Clinical Research Center to prevent alcohol abusers access to alcoholic beverages and to allow BACs in individuals with elevated levels to return to zero. BACs of <10 mg/dl ensured that competitive inhibition of CZX metabolism did not occur. During the overnight stay, volunteers were placed on caffeine-free diets and alcoholics were carefully monitored for any signs of withdrawal. At 7:30 a.m., blood samples were obtained to determine BACs; all subjects possessed values of <5 mg/dl. Blood (320 ml) was drawn at 8:00 a.m. for lymphocyte isolation from all but two alcoholic subjects; in these two cases the large sample was unobtainable. After the blood drawing, 500 mg of CZX was orally administered. Blood samples (10 ml) were subsequently obtained at 0, 0.25, 0.50, 0.75, 1.0, 1.5, 2, 3, 4, 5, 6, 8, 10, and 12 hr. Plasma was separated by centrifugation at 2300 rpm for 15 min at 4°C, immediately frozen, and stored at −20°C until assayed.

Clinical Evaluations.

Subject screening procedures consisted of two types of evaluations. First, subjects completed a survey developed by our laboratory, composed of questions adapted from the CAGE questionnaire, a common screening instrument used by health care professionals to detect alcohol problems (20, 21). Second, a more detailed personal interview was used to compile alcohol usage profiles for all individuals. The profile incorporated physiological, psychological, and social components and was determined by posing questions compiled in the NLAES. The NLAES is an Alcohol Use Disorders and Associated Disabilities Interview Schedule questionnaire similar to that inDiagnostic and Statistical Manual of Mental Disorders, fourth edition, but it was developed primarily for epidemiological research purposes (22). Both surveys were subjectively scored using a protocol developed by an epidemiologist in our laboratory.

Responses to the questionnaire adapted from the CAGE questionnaire were subjectively assigned weighted scores, with greater weight being given to questions that were more specific. The emphasis of the survey was on the frequency of drinking and the amount of alcohol consumed. A simple yes/no question was given less weight than a question specifically asking how frequently the behavior was exhibited. Responses to how often the subject drank alcoholic beverages were scored from 0 to 2, with 0 being never, not in the last year, or less than 1 day/month. A score of 1 was indicative of behavior that occurred either 1–3 days/month or 1 day/week, and a score of 2 indicated that alcohol was consumed 2–3 days/week, 4–5 days/week, or every day. Greater weight was placed on a score of 2 when summing the raw scores for each question.

The NLAES had three variables for each question that dictated the raw score. The first variable determined whether the subject had ever experienced the event or circumstance stated in the question. For an affirmative response a score of 1 was assigned, whereas a negative response was given a score of 0. The second variable assessed whether the experience had occurred in the last 12 months. The third established a weighted factor for how frequently the circumstance had occurred during the last 12 months and ranged from 1 to 5, with 1 being almost never and 5 being all the time. If the event had not happened in the last 12 months, then the weighted score was assigned a value of 0. The score for a given question was equal to the sum of the first and third variables. The second variable (whether the experience had occurred in the last 12 months) was used as an introduction to a more specific investigation of behavior frequency and as an indicator of whether a weighted factor was present. The total score of the survey was the sum of the raw scores for each question. The severity of behavior was quantified by summing the total scores for the questionnaire developed by our laboratory and the NLAES.

Lymphocyte Isolation.

Lymphocytes were immediately separated from 320 ml of whole blood according to the method of Boyum (23). Briefly, 20 ml of Histopaque-1077 (Sigma Chemical Co., St. Louis, MO) was added to a 50-ml conical centrifuge tube. Whole blood was layered onto Histopaque-1077, and tubes were centrifuged at 400g for 30 min at ambient temperature. After centrifugation, the opaque interface was transferred to a new tube, phosphate-buffered saline was added, and tubes were centrifuged at 250g for 10 min at 4°C. Cells were resuspended in RPMI 1640 medium (Gibco BRL Products, Gaithersburg, MD) containing 100 units/ml penicillin, 100 μg/ml streptomycin, and 10% fetal calf serum and were cultured overnight to separate lymphocytes from monocytes. Cultures were then transferred to 50-ml conical tubes and centrifuged at 250g for 10 min at 4°C. The lymphocyte pellet was either resuspended in 5.0 ml of homogenizing buffer (0.1 M Tris-HCl, pH 7.4), containing 0.5 mM phenylmethylsulfonyl fluoride (Sigma), for microsome preparation or mixed with Trizol (Gibco BRL Products) for RNA isolation. Both samples were frozen in liquid nitrogen and stored at −70°C until use.

Microsome Preparation.

Microsomes were isolated from white cells by previously published procedures (16, 24). Briefly, lymphocytes were sonicated on ice for 3 × 60 sec to disrupt cells. Microsomes were then prepared from disrupted cells, and pellets were resuspended in sucrose assay buffer (25). Lymphocyte microsomes were stored at −70°C until use. Protein values were determined with bovine serum albumin as a standard (26).

Immunoblot Analysis.

Microsomal proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose filters. Filters were subsequently blocked in 5% nonfat dry milk/TBST (20 mM Tris buffer, pH 7.6, 137 mM NaCl, 0.1% Tween-20) for 1 hr at 37°C and allowed to react overnight at 4°C in 5% milk/TBST containing a previously characterized anti-human CYP2E1 IgG (5 μg/ml) (27). Filters were then incubated for 60 min with biotinylated goat anti-rabbit IgG (1:2000 in TBST; Calbiochem, La Jolla, CA), followed by a 60-min incubation with streptavidin-conjugated horseradish peroxidase (1:2000 in TBST; Calbiochem) at room temperature. Immunochemical staining was performed by reaction of the filters with 10 ml of enhanced chemiluminescence detection reagents (Amersham, Arlington Heights, IL) for 1 min at room temperature and exposure to Amersham Hyperfilm for 10–30 sec. Immunoreactive CYP2E1 content in lymphocyte microsomes was quantified with a Microtek Scanmaker IIHR scanner interfaced to ImageQuant software. Lymphocyte microsomal protein was applied in various amounts (5–75 μg) to determine the linear range of signal intensity of immunoblots. The concentration of microsomal protein (25 μg) used for all subsequent immunoblot analyses was within the linear portion of that curve.

Reverse Transcription/Polymerase Chain Reaction.

Total RNA from lymphocytes was isolated using Trizol reagent (Gibco BRL Products) and quantified by measurement of its absorbance at 260 nm; purity was assessed by determination of the 260/280-nm ratio. First-strand cDNA synthesis was performed with RNA as previously described (25). The cDNA was then amplified using oligonucleotide primers that were 21 bp in length and flanked CYP2E1 exons 4 (bp 501–523) and 6 (bp 954–976), as described elsewhere (25). The resulting DNA was ligated into pCRII vector (Invitrogen, San Diego, CA) and transformed into competent (INVαF′) Escherichia colicells. Ultrapure plasmid DNA was isolated from the E. colitransformants and sequenced using the dideoxy-chain termination method (28) and Sequenase (United States Biochemicals, Cleveland, OH).

Analytical Methods.

CZX and 6-OH-CZX concentrations in plasma were determined by a reverse-phase HPLC-based assay (9). Plasma samples (250 μl) were thawed on ice, and 750 μl of 2 M sodium acetate buffer, pH 4.5, was added. Each sample was evaluated in triplicate. Twenty microliters (200 activity units) of Helix pomatia type H-2 β-glucuronidase (Sigma) were added to each tube and incubated overnight at 37°C. After incubation, 200 μl of 0.2 M theophylline was added to each tube, for use as an internal standard. The reaction was terminated by addition of 3 ml of 1 N HCl and centrifuged at 3500g for 10 min at 4°C. The supernatant was removed and placed in separate tubes containing 2 ml of ethyl acetate. Samples were vigorously vortex-mixed for 1 min and centrifuged at 2000 rpm for 8 min at 4°C. The organic layer was removed and the aqueous phase was subjected to a second extraction with 2 ml of ethyl acetate. The extracts were pooled and evaporated to dryness under nitrogen with low heat. The residues were resuspended in 100 μl of mobile phase (20%, v/v, acetonitrile/80%, v/v, 0.5% phosphoric acid) and vigorously vortex-mixed. A 20-μl aliquot of extract was subsequently analyzed by use of a model 504 autosampler (Beckman Instruments, Palo Alto, CA), a model 600 solvent delivery system (Waters Millipore, Milford, MA), and a model 486 detector (Waters Millipore) set at 287 nm. Chromatographic separation was achieved with a C₈ Ultrasphere column (5-μm, 4.6 × 250 mm; Beckman Instruments). The mobile phase was pumped at a flow rate of 1 ml/min.

Standard curves of area ratios for CZX/theophylline and 6-OH-CZX/theophylline were constructed from plasma containing various amounts (0.20–8.0 μg/ml) of CZX (Sigma) and 6-OH-CZX (McNeal Consumer Products, Fort Washington, PA). Recoveries of 6-OH-CZX and CZX were 77 and 70%, respectively, with this extraction method. Values for each sample were extrapolated from linear calibration curves (r = 0.99) for the standards. The interday coefficient of variation (N = 22), determined by addition of CZX and 6-OH-CZX to plasma samples, was approximately 15% for each at concentrations of 2, 4, and 8 μg/ml. The limits of detection for both CZX and 6-OH-CZX were 0.09 ± 0.01 μg/ml.

Pharmacokinetic Analysis.

The AUC for CZX and its metabolite were determined using the trapezoid rule. Noncompartmental analysis was used to compare the oral clearance of CZX in control and alcoholic subjects and was determined from the ratio of the administered dose to the total AUC.

Statistical Analysis.

Three separate statistical tests were used to determine significance or possible correlations between study populations. Group comparisons were performed using Student’s t test for independent samples. A one-way analysis of variance was used to determine any statistical significance that might exist between more than two distributions or sample groups. Correlation analyses were also performed, and a curve fit determined the association of the two fields. Statistical calculations were made using GraphPad Instat version 2.05a software. Statistical significance was set at p < 0.05. Values were expressed as the mean ± SE.