Stimulation of mixed-function oxidation of 7-ethoxycoumarin in periportal and pericentral regions of the perfused rat liver by xylitol

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1 Eur. J. Biochem. 237, 1-6 (1983) 0 FEBS 1983 Stimulation of mixed-function oxidation of 7-ethoxycoumarin in periportal and regions of the perfused rat liver by xylitol Steven A. BELINSKY, Frederick C. KAUFFMAN, Sungchul JI, John J. LEMASTERS, and Ronald G. THURMAN Departments of Pharmacology and Anatomy, University of North Carolina at Chapel Hill; and Department of Pharmacology, University of Maryland, Baltimore (Received August 2, 1983) - EJB Rates of 0-deethylation of 7-ethoxycoumarin by perfused livers from fasted, phenobarbital-treated rats were 3.7 pmol x g- x h-l. Approximately 50 % of the product was conjugated. When rates of 7-ethoxycoumarin 0-deethylation were varied by infusing different concentrations of substrate, a good correlation (r = 0.91) was found between rates of 0-deethylation of 7-ethoxycoumarin and fluorescence of 7-hydroxycoumarin detected from the liver surface. Micro-light guides (tip diameter 170 pm) placed on periportal and regions on the liver surface were used to monitor the conversion of nonfluorescent 7-ethoxycoumarin to fluorescent 7-hydroxycoumarin. The 0-deethylation of 7-ethoxycoumarin to 7-hydroxycoumarin increased fluorescence 64 % and 28 % in and periportal regions of the liver lobule, respectively. Rates of 7-ethoxycoumarin 0-deethylation estimated from these increases in fluorescence were 5.2 lmol x g-' x h-' in and 2.2 pmol x g-' x h-' in periportal regions of the liver. During mixed-function oxidation of 7-ethoxycoumarin, the oxidation :reduction state of NADP(H) was similar in both regions of the liver lobule. Xylitol(2 mm) decreased the NADP+/NADPH ratio and stimulated rates of drug metabolism in both regions of the liver lobule. This indicates that conditions exist where the supply of NADPH is an important rate-determining factor for 7-ethoxycoumarin metabolism in both periportal and regions of the liver lobule. Many chemicals (e. g. carbon tetrachloride, acetominophen, bromobenzene) which are activated by the microsomal mixed-function oxidase system produce selective damage to regions of the liver lobule ; however, the mechanisms associated with localized injury within the hepatic lobule remain poorly defined. Since several enzymes, including cytochrome P-450 [l], vary in content across the liver lobule, selective injury to specific zones of the liver may be explained by different rates of mixed-function oxidation across the liver lobule. Immunohistochemical and microspectrophotometric studies indicate that total cytochrome P-450 is greater in than in periportal regions of the liver lobule [1,2]. Inducing agents also have selective effects on different zones of the liver. For example, phenobarbital treatment increases cytochrome P-450 content by more than 100 % in regions while causing only a 50 % increase in periportal areas [2]. In addition to catalytic components of the monooxygenase system, variations in concentrations of cofactors and substrates may also be important determinants of rates of mixedfunction oxidation across the liver lobule. While the rate of reduction of components of the mixedfunction oxidase system is rate-limiting for xenobiotic metabolism in isolated microsomes [3] in the presence of excess cofactor, previous reports from this laboratory and others have shown that the supply of NADPH is an important determinant of rates of mixed-function oxidation in the whole liver [4-71. Thus, differences in the capacity to generate NADPH in periportal and regions of the liver may account for variations in rates of monooxygenation across the liver lobule. Using quantitative cytochemistry, glucose-6-phosphate dehydrogenase activity was shown to be twice as high in as in periportal regions of the liver lobule [8]. In addition, pretreatment with phenobarbital increased glucose- 6-phosphate dehydrogenase activity to a greater extent in than in periportal hepatocytes. Although measure ments of cytochrome P-450 distribution and glucosed-phosphate dehydrogenase activity indicate that the maximal capacity to metabolize drugs may vary greatly across the liver lobule, the respective roles of NADPH generating enzymes and microsomal components in regulating mixed-function oxidation in different regions of the liver lobule under physiological conditions is not clear. Recently, Ji et al. [9] developed a method to determine rates of 7-ethoxycoumarin 0-deethylation in periportal and regions of the liver lobule by measuring the fluorescence of 7-hydroxycoumarin produced via mixed-function oxidation of nonfluorescent 7-ethoxycoumarin. Maximal rates of 7-hydroxycoumarin formation in livers from fed, phenobarbital-treated rats were about twofold higher in than in periportal regions [9]. This study was initiated to determine the effect of experimentally altered NADPH supply on mixed-function oxidation in periportal or regions of the liver lobule. Xylitol and sorbitol were used to increase the supply of NADPH and ethanol was used to elevate NADH and decrease NADPH [lo]. The sugars stimulated 7-ethoxycoumarin 0-deethylation equally in both regions of the liver lobule, indicating that NADPH is an important rate-determinant for mixed-function oxidation in periportal and regions of the liver lobule. MATERIALS AND METHODS Animals Female Sprague-Dawley rats, g, were treated with sodium phenobarbital (1 mg/ml) in drinking water for at

2 2 least one week prior to use [l I]. Fasted animals were deprived of food for 24 h prior to use and were employed in this study because xylitol stimulated mixed-function oxidation to a much greater extent in livers from fasted than fed rats [12]. Liver perfusion Details of the perfusion technique have been described elsewhere [4]. Livers were perfused at 37 "C with Krebs- Henseleit bicarbonate buffer, ph 7.4, saturated with an oxygen/carbon dioxide mixture (95 : 5) in a nonrecirculating system. The fluid was pumped into the liver via a cannula placed in the portal vein and flowed past a teflon-shielded, Clark-type oxygen electrode. Rates of oxygen uptake were calculated from the influent minus effluent oxygen concentration difference, the flow rate and the liver wet weight. A stock solution of 7-ethoxycoumarin was dissolved in N,N,-dimethylformamide and added to Krebs-Henseleit bicarbonate buffer to give a final concentration of 35 mm N,N-dimethylformamide and 100 pm 7-ethoxycoumarin when infused into the liver. Under these conditions, N,N-dimethylformamide had no effect on mixed-function oxidation. Following the addition of 7-ethoxycoumarin, effluent samples were collected every 2 min to determine the concentration of free and conjugated 7- hydroxycoumarin. The concentration of 7-hydroxycoumarin was measured fluorometrically ( nm) using an Eppendorf fluorometer. The total rate of 7-hydroxycoumarin production was calculated from the sum of free and conjugated 7-hydroxycoumarin produced by the 0-deethylation of 7- ethoxycoumarin, the flow rate and the liver wet weight. Determination of conjugates of 7-hydroxycoumarin Sulfate and glucuronide conjugates of 7-hydroxycoumarin were determined by measuring 7-hydroxycoumarin formed after incubation of 1.0-ml samples of perfusate with 0.5 ml of 180 mm potassium phosphate buffer, ph 7.4, containing 250 units of purified b-glucuronidase and 25 units of sulfatase activity (Sigma), respectively, for 1.5 h at room temperature. This procedure hydrolyzed over 95 % of all glucuronide and sulfate conjugates of 7-hydroxycoumarin. Measurement of 7-hydroxycoumarin fluorescence from periportal and regions of the liver Rates of mixed-function oxidation in the two zones of the liver were measured with the method first reported by Ji et al. [9,13]. Pairs of micro-light guides constructed from strands of 70-pm-diameter glass optical fibers were placed siniultaneously on periportal and regions on the surface of the perfused liver. Anterograde (via portal vein) and retrograde (via the vena cava) perfusions of liver with India ink identified lightly pigmented regions as periportal areas and darkly pigmented spots as regions (Fig. 1). Fluorescence due to 7-hydroxycoumarin formation from 7-ethoxycoumarin was measured on periportal and regions of the liver by illuminating tissue with light at 366 nm and measuring fluorescence at 450 nm. The lower basal fluorescence in the area relative to the periportal region (Fig. 4; Table 2) is probably due to quenching of pyridine nucleotide fluorescence by higher concentrations of cytochrome P-450 in the area [9]. Experiments were therefore performed to evaluate the effect of this fluorescence Fig. 1. Light micrograph of the.rurfuce qf'fhe hemoglobin:free perfused rut liver. The liver has a natural mottled pigmentation. Photograph was prepared with epi-illumination. Lightly pigmented areas correspond to periportal regions while the darkly pigmented spots are (30X magnification ; horizontal bar = 500 pm) quenching at various 7-hydroxycoumarin concentrations. In the presence of N, and 20 mm ethanol, 7-hydroxycoumarin was not conjugated by the liver (Conway, Kauffman and Thurman, unpublished). Under these conditions, the ratio of the fluorescence signals from periportal/ areas was essentially constant ( ) over a wide range of 7-hydroxycoumarin concentrations (0-120 pm). Moreover, similar ratios in basal fluorescence were observed under normoxic conditions. Therefore, all fluorescence signals were expressed as a percentage of basal fluorescence to estimate 7-hydroxycoumarin concentrations properly in tissue in the various regions of the liver lobule. Under the conditions employed, 7- ethoxycoumdrin was practically nonfluorescent. Although the wavelengths employed for detection of 7-hydroxycoumarin fluorescence from the tissue also excite NADH and NADPH, fluorescence due to pyridine nucleotides does not interfere because 7-hydroxycoumarin is 200-times more fluorescent than pyridine nucleotides on a molar basis (unpublished results). Rates of 7-ethoxycoumurin 0-deethylution in periportal and regions of the liver lobule Rates of mixed-function oxidation of 7-ethoxycoumarin were calculated from 7-hydroxycoumarin detected in the effluent perfusate and were compared with increases in 7-hydroxycoumarin fluorescence from the liver surface measured with a large-tipped light guide (2-mm diameter). Rates of mixed-function oxidation were varied by infusing different concentrations of 7-ethoxycoumarin. A good correlation between fluorescence of 7-hydroxycoumarin and the O-deethylation of 7-ethoxycoumarin has been observed previously in livers from fed rats [9]. Similar data were obtained in this study with fasted rats (data not shown). This correlation allows us to convert fluorescence readings measured with the micro-light guide into local rates of mixed-function oxidation.

3 3 To estimate local rates of 7-ethoxycoumarin O-deethylation in periportal and regions, micro-light guides were placed on light and dark areas as described above. During the metabolism of 7-ethoxycoumarin, fluorescence changes, as a percentage of basal fluorescence, were determined in periportal and regions. From the fluorescence changes arising from 7-hydroxycoumarin formation and from rates of mixed-function by the entire liver, local rates of 7-hydroxycoumarin formation in the two zones of the liver lobule were calculated [9]. Measurement of pyridine nucleotides in periportal and regions of the liver lobule The left lateral lobe of the liver was frozen during perfusion by pressing an aluminum mallet chilled in liquid nitrogen gently on the lobe and then immersing the liver in liquid nitrogen. This process resulted in very rapid freezing of the liver surface without disruption of the lobular structure of the liver. Blocks of tissue approximately 3 mm square were dissected in the frozen state from the portion of liver frozen with the mallet. The blocks were mounted on a cryostat chuck and cut into 20-pm sections at -15 "C. The frozen sections were lyophilized at -40 "C to maintain histological structure [14,15] and facilitate visualization of periportal and regions (Fig. 2). Both zones were microdissected and weighed on a quartz fiber balance [14]. Samples of the two regions taken for analyses weighed pg. Total NADP' and NADPH were measured in alkaline extracts of the microdissected samples employing enzymatic cycling procedures [15]. NADPH was measured in extracts which were heated 15 min at 60 "C to destroy NADP' [35]. RESULTS ESfect of xylitol on 7-ethoxycoumarin 0-deethylation Rates of 7-ethoxycoumarin 0-deethylation were about 3.7 pmol x g-' x h-' in livers from fasted, phenobarbitaltreated rats (Fig. 3, Table 1). Approximately 50 % of the product formed was conjugated as either the glucuronide or sulfate (Table 1). Infusion of xylitol (2 mm) doubled rates of mixed-function oxidation and conjugation (Fig. 3, Table 1). Effect of xylitol on 7-hydroxycoumarin fluorescence from periportal and regions of the liver Micro-light guides placed on periportal and regions of the liver lobule were used to monitor changes in fluorescence when 7-ethoxycoumarin (100 pm) was infused in the anterograde direction. Increases in maximal fluorescence expressed as a percentage of basal fluorescence were more than twice as great in as in periportal regions of the liver lobule (Fig. 4, Table 2). Subsequent infusion of 2 mm xylitol produced further increases in fluorescence in both periportal and regions of the liver (Fig. 4, Table 2). Minimal changes in fluorescence due to xylitol alone (Fig. 4) were taken into account in subsequent calculations. With anterograde perfusions, 7-ethoxycoumarin reaching the regions may be less than that delivered to the periportal zones. Thus, to compare rates of metabolism with equivalent substrate concentrations in the two regions, we also performed retrograde perfusions. When 7-ethoxycoumarin Fig. 2. Light microxraph of freeze-dried tissue sections from hernoglohin:fi.eeperfu,used rat liver. Livers were frozen during perfusion using an aluminum mallet chilled in liquid nitrogen to preserve the structural heterogeneity of the liver. Sections (20 pm) were cut from the frozen liver and lyophilized at -40 "C to preserve histological structure. Periportal (light areas) and (dark areas) regions are easily visible (24X magnification, horizontal bar = 400 pm) c._ 0 100pM 7-ethoxycoumarin total 7-hydroxycournarin free 7-hydroxy ?- cournarin 0 x o w I r. Minutes of perfusion Fig. 3. Elfect ofxylitol on rates of drug metabolism and conjugation. 7-Ethoxycoumarin (100 pm) and xylitol (2 mm) were infused into livers from fasted, phenobarbital-treated rats at the times designated by the horizontal bars and vertical arrows. Rates of free and conjugated 7-hydroxycoumarin formation were calculated as described in Materials and Methods Table 1. Effect of xylitol on 7-ethoxycoumarin 0-deethylution in livers fiom fasted phenobarbital-treated rats Data represent maximal rates of 7-hydroxycoumarin production from 7-ethoxycoumarin in the presence or absence of xylitol. Values are means f SEM from eight livers Xylitol mm Ethoxycoumarin 0-deethylation free conjugated total pmol x g-' x h-' 1.8k f 0.4" 3.8f0.3a 7.5 L-0.5" P<O.OOI with respect to control values.

4 4 loopm 7-ethoxycournarin 1 Fl 2mM I j xygtoi p LO Minutes of perfusion Fig Hydroxycoumarin fluorescence from tissue following the addition of 7-ethoxycournurin and xylitol. Two micro-light guides (tip diameter= 170 pm) were placed on two adjacent periportal regions (1-3 mm apart) on the left lateral lobe of the liver. The output voltages ( V) of the photomultipliers were adjusted to give similar anode currents in both channels. Subsequently, one micro-light guide was moved to a area. 7-Ethoxycoumarin (100 pm) and xylitol (2 mm) were then infused at the times designated by the horizontal bars and vertical arrows. 7-Hydroxycoumarin fluorescence from the liver surface was determined as described in Materials and Methods Table 2. Effect of xylitol on rates of mixed-function oxidation of 7-ethoxycoumarin in periportal andpericentrul regions of the liver lobule in livers from fasted phenobarbital-treated rats Experiments were carried out as described in legends to Fig. 3 and 4. The lower basal fluorescence in the area relative to the periportal region is probably due to fluorescence quenching by higher concentrations of cytochrome P-450 in the area. The anode current (na) from the photomultiplier (EM1 type 9824B) was measured at an output voltage of OV. Increases in tissue fluorescence and local rates of 7-ethoxycoumarin 0-deethylation following addition of xylitol were corrected for the small increase in NADH fluorescence observed with xylitol alone (see Fig. 4). Data represents mean SEM, from five livers Parameter Value in lobular region periportal Basal tissue fluorescence ( nm) (na) 31.8k Ifr 0.7 Tissue fluorescence increase (% basal fluorescence) upon infusion of 7-ethoxycoumarin 28.4k k4.6 Local rate of 7-ethoxycoumarin 0-deethylation (pmol x g-' x h-i) 2.2k k0.7 Tissue fluorescence increase (% basal fluorescence) following addition of xylitol during 7-ethoxycoumarin 0-deethylation 38.6 k 5.6" k 7.0b Local rate of 7-ethoxycoumarin 0-deethylation following addition of xylitol (pmol x g-' x h-l) 3.9k0.3" " a P<O.O5 with respect to control values. P<O.Ol with respect to control values. of xylitol (2 mm) stimulated rates of 7-ethoxycoumarin 0- deethylation significantly in both regions (Table 2). Measurement of NADP(H) in periportal and regions of the liver Levels of NADP' and NADPH were identical in periportal and regions of the liver lobule both in the presence and absence of 7-ethoxycoumarin (Table 3). The addition of 7-ethoxycoumarin, as in earlier experiments with hexobarbital[16], caused a small increase in the NADP' pool size which was accompanied by an increase in the NADP+/NADPH ratio in both regions of the liver lobule (Table 3). When xylitol was infused with 7-ethoxycoumarin, an increase in NADPH content and a decrease in the NADP+/NADPH ratio was observed in both regions (Table 3). Effect of sorbitol and ethanol The addition of xylitol to the perfused liver caused an increase in cellular NADH [17] as well as NADPH (Table 3). Thus, it is possible that the stimulation of 7-hydroxycoumarin production by xylitol could result from NADH synergism of NADPH-dependent drug metabolism [18,19]. To test this hypothesis, sorbitol, a carbohydrate which increases NADPH and NADH content [lo] and ethanol, which elevates NADH but lowers NADPH [lo], were infused into the perfused liver during the metabolism of 7-ethoxycoumarin. Sorbitol stimulated 7-hydroxycoumarin production by % in both regions of the liver lobule (Table4). In contrast, ethanol inhibited 7-hydroxycoumarin production, but the effect was confined largely to periportal regions of the liver lobule. was infused in the retrograde direction, fluorescence of 7-hydroxycoumarin was also more than twice as great in than periportal regions of the liver. Under these conditions, xylitol also increased fluorescence in both regions of the liver to the same extent as with anterograde infusion (data not shown). Local rates of 7-ethoxycoumarin 0-deethylation calculated from fluorescence changes were approximately twice as great in as periportal regions of the liver. Infusion DISCUSSION In livers from fasted, phenobarbital-treated rats, rates of 7-hydroxycoumarin production were 2.2 and 5.2 Fmol x g-' x h-' in periportal and regions of the liver lobule, respectively (Table 2). The purpose of this study was to determine whether these differences in rates of mixed-function oxidation could be explained by differences in NADPH supply or in cytochrome P-450 in various zones of the liver lobule.

5 Table 3. Effect of xylitol on pyridine nucleotides in periportal and regions of livers from fasted, phenobarbital-treated ruts Surface of livers from fasted, phenobarbital-treated rats were frozen with an aluminum mallet at liquid nitrogen temperature after 36 min of perfusion. 7-Ethoxycoumarin (100 pm) and xylitol (2 mm) infusions were initiated after 20min and 30min of perfusion, respectively. Five to seven samples of periportal or areas were sampled from each liver for determination of pyridine nucleotides. Metabolite concentrations were measured in freeze-clamped livers as described in Materials and Methods. Values are means + SEM from five livers 5 Additions Pyridine nucleotides in lobular region periportal NADP' NADPH total NADP"/NADPH NADP' NADPH total NADP' /NADPH NADP NADP nrnol/mg dry liver nmol/mg dry liver None (control) 1.OkO.1 0.5k k f k k k k Ethoxycoumarin 1.4 k t ko k k k k k Ethoxycoumarin and xylitol 0.9k0.3" 1.0k0.3" 1.9k k0.93" 1.2k k k " a P < 0.05 with respect to 7-ethoxycoumarin addition. Table 4. Effect of sorbitol andethanolonperiportal and rates of 7-hydroxycoumarin production in livers from fasted, phenobarbitaltreated ruts Experiments were carried out essentially as described in legends to Fig. 3 and 4 with the exception that sorbitol (2mM) or ethanol (20 mm) were infused into the liver instead of xylitol. Data represents meanfsem; n=number of livers Addition n 7-Hydroxycoumarin production in lobular region periportal pmol x g-' x h-' None k k mm sorbitol 5 3.7k0.6" 7.4 k 0.8" 20 mm ethanol 6 1.0k0.2" 3.8 k0.3 a P<O.OI with respect to no addition. Although NADPH content was similar in both periportal and regions (Table 3), turnover of this cofactor could differ considerably. If the liver could not regenerate NADPH, rates of 7-hydroxycoumarin formation observed in these studies could be sustained for only 3 min (Tables 2 and 3). Thus, it is apparent that the liver must constantly resynthesize NADPH at high rates. If rates of mixed-function oxidation are regulated predominantly by the concentration of cytochrome P-450, which is greater in hepatocytes [l], then an increase in NADPH content in the liver should have little effect on local rates of 7-hydroxycoumarin formation. However, addition of xylitol enhanced rates of 7-hydroxycoumarin production from 7-ethoxycoumarin (Fig. 3, Table 1) in both periportal and regions of the liver lobule (Fig. 4, Table 2). This action of xylitol is best explained by an increase in NADPH supply. This conclusion is supported by the observation that sorbitol, which increases NADPH supply, also stimulates mixed-function oxidation while ethanol, which increases NADH but decreases NADPH, produced inhibition (Table 4). In mammalian liver, xylitol is oxidized predominantly by an NAD +-linked dehydrogenase to form xylulose which is subsequently phosphorylated and enters intermediary metabolism at the triose phosphate level [IA. In livers from fasted rats, xylitol is converted rapidly into glucose [20,21], which is subsequently phosphorylated to glucose 6-phosphate and generates NADPH for mixed-function oxidation via the pentose phosphate shunt. Furthermore, 6-aminonicotinamide, a potent inhibitor of the pentose phosphate shunt, abolished the stimulation of mixed-function oxidation by sorbitol, a sugar that is metabolized via the same reaction sequence as xylitol [lo]. In the present study, xylitol caused a significant decrease in the NADP+/NADPH ratio in both regions of the liver lobule. It also stimulated 7-ethoxycoumarin metabolism in both periportal and regions (Table 3). Thus, it is reasonable to conclude that NADPH supply is a major determinant of rates of mixed-function oxidation in both regions of the liver lobule. Since NADPH supply is an important rate determinant of mixed-function oxidation, it could account for differences in rates observed in periportal and regions of the liver lobule (Table 2, Fig. 4). For example, it is possible that NADPH utilization for lipid or cholesterol biosynthesis predominates in periportal cells allowing less NADPH to be utilized for mixed-function oxidation. In fact, Smith and Wills [8] observed greater lipid synthesis in periportal than hepatocytes. On the other hand, Brunengraber (personal communication) observed little lipid synthesis in the substratefree perfused rat liver. Another possibility is that the supply or turnover of NADPH varies across the liver lobule as a consequence of the heterogenous distribution of enzymes that generate NADPH [8,22]. Based on the observation by Smith and Wills [8] that glucose-6-phosphate dehydrogenase activity was four-times greater in than in periportal hepatocytes of phenobarbital-treated rats, one might argue that enhanced pentose phosphate shunt activity is responsible for higher rates of mixed-function oxidation in areas of the liver lobule (Table 2). However, our experiments were performed in livers from fasted rats where carbon flux over the pentose phosphate pathway is reduced severely [23] due to limiting amounts of the substrate glucose 6-phosphate.

6 6 Differences in NADPH generation via mitochondria1 oxidations could also explain the differences in local rates of mixedfunction oxidation observed in this study. Cytochrome P-450, at least in livers from phenobarbitaltreated rats, is distributed unevenly across the liver lobule [I]. The cytochrome P-450s induced by phenobarbital are greater in than in periportal regions, whereas the cytochrome P-450 induced by 3-methylcholanthrene is distributed evenly across the liver lobule [l]. The highest rate of turnover with 7-ethoxycoumarin as substrate occurs with the methylcholanthrene-induced isoenzyme [24,25] ; however, a substantial amount of this substrate is also metabolized by the isoenzyme induced by phenobarbital. In livers from phenobarbital-treated rats the concentration of the phenobarbitalinduced isoenzyme is 16-fold greater than that induced by methylcholanthrene [26]. Thus, the greater rate of metabolism of 7-ethoxycoumarin in these experiments is probably due to the phenobarbital-inducible form of cytochrome P-450. Thus, differences in the distribution of the latter isoenzyme across the liver lobule could also contribute to the observed differences in rates of 7-hydroxycoumarin formation in zones and periportal regions of the liver (Table 2). In these studies, evidence has been presented indicating that NADPH supply is a major rate determinant of the mixedfunction oxidation of 7-ethoxycoumarin in both periportal and regions of the liver lobule. However, NADPH supply is not the sole determinant of rates of mixed-function oxidation. Differences in mixed-function oxidation across the liver lobule may also involve the uneven distribution of cytochrome P-450 across the liver lobule. Since the P-450 -CO complex increased upon the addition of hexobarbital in livers from fed, phenobarbital-treated rats, Sies and Brauser concluded that NADPH supply was not ratelimiting in livers from fed, phenobarbital-treated rats [27]. It would be interesting, therefore, to identify factors responsible for the metabolism of drugs other than 7-ethoxycoumarin in periportal and regions of the liver in various nutritional states. Supported, in part, by grants CA and CA from the National Cancer Institute and from the American Heart Association. R.G.T. was the recipient of Research Scientist Career Development Award AA S.A.B. is supported by a predoctoral traineeship ES from the National Institutes for Education, Health and Science. REFERENCES 1. Baron, J., Redick, J. A. & Guengerich, P. F. (1978) Life Sci. 23, Gooding, P. E., Chayen, J., Sawyer, B. & Slater, T. F. (1978) Chem. Biol. Inter. 20, Imai, Y., Sato, R. & Iyanagi, T. (1977) J. Biochem. (Tokyoj 82, Thurman, R. G., Marazzo, D. P., Jones, L. S. & Kauffman, F. C. (1977) J. Pharmacol. Exp. Ther. 201, Reinke, L. A,, Kauffman, F. C., Belinsky, S. A. & Thurman, R. G. (1979) J. Pharmacol. Exp. Ther. 213, Moldeus, P., Grundin, R., Vadi, H. & Orrenius, S. (1974) Eur. J. Biochem. 46, Belinsky, S. A., Reinke, L. A,, Kauffman, F. C. & Thurman, R. G. (1980) Arch. Biochem. Biophys. 204, Smith, M. T. &Wills, E. D. (1981) Biochem. J. 200, Ji, S., Lemasters, J. J. & Thurman, R. G. (1981) Mol. Pliarmacol. 19, Reinke, L. A., Belinsky, S. A., Kauffman, F. C., Evans, R. K. & Thurman, R. G. (1982) Biochem. Pharmacol. 31, Marshall, W. S. & McLean, A. E. (1968) Biochem. Pharmacol. 18, Reinke, L. A., Kauffman, F. C. & Thurman, R. G. (1980) Biochem. Pharmacol. 29, Ji, S., Lemasters, J. J. & Thurman, R. G. (1980) FEBS Lett. 113, ; Corrigenda (1980) FEBS Lett. 114, Lowry, 0. H. (1953) J. Histochem. Cytochem. 1, Lowry, 0. H. & Passonneau, J. V. (1972) A Flexible System of Enzymatic Analysis, pp , Academic Press, New York. 16. Sies, H. & Kandel, M. (1970) FEBS Lett. 9, Jakob, A., Williamson, J. R. & Asakura, T. (1971) J. Biol. Chem. 246, Hildebrandt, A. & Estabrook, R. W. (1971) Arch. Biochem. Biophys. 143, Correia, M. A. & Mannering, G. J. (1973) Drug Metab. Dispos. I, Ross, B. D., Hems, R. & Krebs, H. A. (1967) Biochem. J. 102, Woods, H. F. & Krebs, H. A. (1973) Biochem. J. 134, Jungermann, K. & Katz, N. (1982) Hepatology, 2, Belinsky, S. A., Reinke, L. A., Kauffman, F. C., Scholz, R. & Thurman, R. G. (1981) Fed. Proc. 40, Thomas, P. E., Reik, L. M., Ryan, D. E. & Levin, W. (1981) J. Biol. Chem. 256, Ryan, D. E., Thomas, D. E., Korzeniowski, D. & Levin, W. (1979) J. Biol. Chem. 254, Dannan, G. A,, Guengerich, F. P., Kaminsky, 0. S. & Aust, S. 0. (1983) J. Biol. Chem. 258, Sies, H. & Brauser, B. (1971) Eur. J. Biochem. 15, R. G. Thurman, S. Belinsky, and S. Ji, Department of Pharmacology, School of Medicine of the University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA J. Lemasters, Department of Anatomy, School of Medicine of the University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA F. C. Kauffman, Department of Pharmacology, University of Maryland, Baltimore, Maryland, USA 21201