Introduction

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Theophylline is a bronchodilator that is frequently used in the treatment of asthma and chronic obstructive pulmonary disease (Murciano et al., 1984, 1989). Because the therapeutic range of theophylline is reportedly narrow (10-20 µg/ml), therapeutic drug monitoring is essential. The theophylline dosage required to achieve therapeutic concentrations varies among subjects, largely because of differences in metabolism (Boobis et al., 1991). Another pharmacokinetic determinate is protein binding (Shaw et al., 1982; Du Souich et al., 1993). Theophylline binds mainly to albumin, with protein binding of approximately 50% (Buss et al., 1983). Hypoalbuminemia has been found to affect the protein binding of theophylline in some situations. Decreased theophylline binding and physiological hypoalbuminemia have been observed in the third trimester of pregnancy (Dean et al., 1980; Connelly et al., 1990). For patients with poor glycemic control of insulin-dependent diabetes mellitus, decreases in serum albumin levels are accompanied by increases in the free fraction of theophylline (Karrapati et al., 1995). This increased free fraction of theophylline with hypoalbuminemia implies an effect on pharmacokinetic parameters such as V_d¹ and/or theophylline CL (Karrapati et al., 1995).

We established a mutant strain of HLRs, which were bred from SDRs (Watanabe et al., 1996; Nakura et al., 1997). Reduction of P450 cholesterol 7-hydroxylase (CYP7A1) activity in HLRs may contribute to a decrease in cholesterol elimination, resulting in increased serum cholesterol levels (Brassil et al., 1996). Because HLRs show extremely low serum albumin concentrations, compared with SDRs, we examined the effect of hypoalbuminemia on theophylline disposition using HLRs. This study provides instructive information concerning the pharmacokinetics for theophylline treatment of patients with hypoalbuminemia.

	Materials and Methods

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Chemicals. Theophylline, 7-hydroxycoumarin, 3,3'-diaminobenzidine, and benzo[a]pyrene were obtained from Wako Pure Chemicals (Tokyo, Japan). Glucose-6-phosphate, glucose-6-phosphate dehydrogenase, and NADP⁺ were purchased from Oriental Yeast (Tokyo, Japan), and 7-ethoxycoumarin was purchased from Aldrich Chemical Co. (Milwaukee, WI).

Determination of Theophylline Pharmacokinetics. HLRs, exhibiting 3-fold higher serum cholesterol levels than do original SDRs, have been raised and bred in our laboratory for more than 60 generations. Eight-week-old male SDRs and HLRs weighing 219 to 242 g were raised in our breeding colony after brother-sister mating. These rats were maintained in air-conditioned quarters with 12-hr light/dark cycles and were given laboratory chow (CE-2; Clea Japan, Tokyo, Japan) and water ad libitum. Theophylline (10 mg/kg) dissolved in saline was injected into the tail veins of SDRs (N = 5) and HLRs (N = 5); blood samples (300 µl) were collected at 5 and 30 min and 1, 2, 3, 6, and 10 hr after injection. The serum theophylline concentrations were measured by an immunofluorescence method using a TDX^® system (Dynabot, Tokyo, Japan). Pharmacokinetic parameters were calculated using noncompartmental methods. The slope of the terminal elimination phase () was obtained by least-squares linear regression analysis. The elimination t_1/2 was calculated as ln 2/. The AUC was calculated with the trapezoidal rule. The values were extrapolated to infinity by dividing the last measured plasma concentration by . CL and V_d were calculated as CL = dose/AUC and V_d = CL/, respectively. For a second set of HLRs (N = 5) and SDRs (N = 5), which were used to measure the free fraction of theophylline, the same dose was administered iv and blood samples were collected at 1, 2, 3, and 6 hr after the injection. The unbound theophylline concentrations were measured after ultrafiltration using Ultrafree-MC filters (Millipore Corp., Bedford, MA). The pH values of these serum specimens varied from 7.8 to 8.0. Serum albumin concentrations were measured using a kit (Albumin B-Test Wako; Wako Pure Chemicals).

Determination of Enzymatic Activities in Liver Microsomes. Eight-week-old male SDRs and HLRs weighing 230-254 g were killed by decapitation in the morning; the livers were removed and homogenized with 3 volumes of ice-cold 1.15% potassium chloride. Liver microsomes were prepared by sequential centrifugation at 9000g for 20 min and then at 105,000g for 60 min. The amounts of microsomal protein were determined by the method of Lowry et al. (1951), using bovine serum albumin as the standard. A typical incubation mixture for the assay of ECOD and AHH activities consisted of 100 mM sodium/potassium phosphate buffer (pH 7.4), 0.05 mM EDTA, 0.5 mg of microsomal protein, an NADPH-generating system (5 mM magnesium chloride, 0.5 mM NADP⁺, 5 mM glucose-6-phosphate, and 1 unit of glucose-6-phosphate dehydrogenase), and a substrate (0.5 mM ethoxycoumarin or 3.2 mM benzo[a]pyrene), in a final volume of 1 ml. The reaction was started by the addition of the NADPH-generating system. After a 2-min preincubation, the reaction mixture was incubated at 37°C for 10 min and 5 min for ECOD and AHH, respectively. One milliliter of 5% trichloroacetic acid (for ECOD) or 1 ml of acetone (for AHH) was added to the mixture to stop the reaction. The activity of ECOD was estimated by determination of 7-hydroxycoumarin concentrations (Aitio, 1978), and AHH activity was measured by determination of the concentrations of the hydroxylated metabolites of benzo[a]pyrene (Nebert, 1978). The activities of both enzymes were measured spectrophotofluorometrically (excitation/emission wavelengths of 380/460 nm and 396/518 nm for ECOD and AHH, respectively).

Immunoblot Analysis of CYP1A1/1A2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed according to the methods of Laemmli (1970) and Guengerich et al. (1982), with a 3% stacking gel and a 10% separating gel (gel size, 13 × 10 cm), and electrophoresis was conducted at 30 mA for 2 hr. Western blot analysis was performed with a polyclonal antibody specific to rat CYP1A1/1A2 (Daiichi Pure Chemicals Co., Ltd., Tokyo, Japan). The bands were detected by the developed peroxidase reaction (Vectastain ABC kit; Vector Laboratories, Burlingame, CA), with diaminobenzidine as a substrate for peroxidase.

Statistical Analysis. Data are presented as mean ± SE. Data were compared using an unpaired t test. A p value of <0.05 was considered significant.

	Results

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The HLRs had significantly lower albumin concentrations than did the SDRs (2.0 ± 0.05 and 4.1 ± 0.03 mg/dl, respectively). No significant differences were seen between the HLRs and SDRs in body or liver weight (table 1).

Serum concentration-time curves for theophylline in HLRs and SDRs are shown in fig. 1, and the pharmacokinetic parameters are summarized in table 2. The AUC from 0 to 10 hr was significantly lower for the HLRs than for the SDRs. Conversely, the CL was significantly greater in the HLRs than in the SDRs. The theophylline V_d was significantly increased in the HLRs, compared with the SDRs. No significant difference was noted between the HLRs and the SDRs in the theophylline t_1/2.

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Concentration-time profiles for theophylline in SDRs and HLRs. Each point with error bar represents the mean ± SE from five SDRs () or HLRs ().

The theophylline free concentration in the SDRs was approximately 30% of the total concentration at each sampling point. In contrast, the unbound theophylline concentration in HLRs was approximately equal to the total concentration. The bound fraction of theophylline was lower in the HLRs than in the SDRs (fig. 2).

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Concentration-time profiles for free and total theophylline in SDRs and HLRs. Each point with error bar represents the mean ± SE, from five rats, for total theophylline concentration () or free fraction () in the serum.

Table 3 shows the P450 contents and the ECOD and AHH activities. The P450 content in HLR liver microsomes was four fifths of that in SDR liver microsomes. ECOD and AHH activities in HLR liver microsomes were 78 and 59%, respectively, of the activities in SDR liver microsomes. The CYP1A1/1A2 apoprotein levels were determined by immunoblot analysis, and the CYP1A1/1A2 apoprotein levels in HLRs were 57% of those found in SDRs (fig. 3).

View larger version (53K): [in this window] [in a new window]   Fig. 3.   Expression levels of CYP1A1/1A2 protein in SDRs and HLRs, detected by Western blot analysis. The amounts of microsomal protein applied in each lane were 50 µg.

	Discussion

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In the present study, we demonstrated the effect of hypoalbuminemia on theophylline pharmacokinetics, with HLRs serving as the animal model because their serum albumin and theophylline protein binding levels were significantly lower than those found in SDRs. Theophylline is metabolized by 8-hydroxylation to 1,3-dimethyluric acid, which accounts for about one half of the CL of the drug in humans (Ogilvie, 1978), and by N-demethylation to 3-methylxanthine and 1-methylxanthine. Although the former reaction is catalyzed by several P450 subfamilies (Zhang and Kaminsky, 1995; Sarkar et al., 1991), CYP1A2 is reported to play a major role at lower substrate concentrations (Zhang and Kaminsky, 1995). CYP1A is also responsible for the latter reaction (Sarkar et al., 1991; Sarkar and Jackson, 1994).

The increase in theophylline CL cannot be explained by the observed lower levels of CYP1A1/1A2 apoprotein and activities in HLRs, compared with those in SDRs. It could be related to the increase in the theophylline V_d. The lack of a significant difference in the theophylline t_1/2 between the HLRs and the SDRs might support this assumption that the increase in CL resulted from an increase in the theophylline V_d. An increase in the unbound fraction might contribute to an increase in the theophylline V_d. A linear relationship between the propranolol V_d and the plasma unbound fraction was reported by Branch et al. (1976), which agrees with the results of this study. In our study, the theophylline V_d was increased and the serum concentration was reduced in rats with hypoalbuminemia, in comparison with the theophylline V_d and serum concentration in rats with normal albumin levels.

Karrapati et al. (1995) reported a positive correlation between hemoglobin A1c levels and plasma theophylline CL in patients with insulin-dependent diabetes mellitus, and they suggested that patients with poor glycemic control have higher theophylline CL values. Those authors also observed lower serum albumin concentrations and higher theophylline free fractions in those patients. In patients with poor glycemic control, an increase in the V_d caused by hypoalbuminemia might contribute to an increase in theophylline CL.

The HLRs exhibited higher serum cholesterol and triglyceride concentrations, compared with the SDRs. Because hyperlipidemia reportedly does not affect theophylline pharmacokinetics in rabbits (Wojcicki et al., 1996), the difference in theophylline pharmacokinetics between SDRs and HLRs might not be related to the higher serum cholesterol or triglyceride concentrations in HLRs.

In summary, the rats with hypoalbuminemia had significantly greater V_d values, which resulted in an increase in the CL, although the rats had lower CYP1A1/1A2 activity and expression than did the rats with normoalbuminemia. Hypoalbuminemia is associated with a number of diseases and disorders (Steinfeld, 1964; Toporovski et al., 1982). Patients with asthma or chronic obstructive pulmonary disease may develop hypoalbuminemia. It is hoped that our current results will inform further investigations concerning theophylline pharmacokinetics in patients with hypoalbuminemia. Further study is required to demonstrate the effects of an increase in the unbound fraction on the theophylline pharmacodynamics with hypoalbuminemia.