Alvin N. Kotake

The caffeine CO2 breath test: Dose response and route of N‐demethylation in smokers and nonsmokers

The optimal conditions for performing the caffeine CO2 breath test (CBT) were investigated in smokers and nonsmokers. Caffeine labeled with13C or 14C in all three (1, 3, and 7) methyl groups or specifically in the 1‐, 3‐, or 7‐methyl groups were orally administered to healthy adults and the expiration of labeled CO2 was measured for 8 or 24 hr. The absolute rate of labeled CO2 excretion from trilabeled caffeine was proportional to the dose up to 3 mg/kg in all subjects. In smokers, the rate of labeled CO2 excretion averaged twice that in nonsmokers at all doses. A correlation was observed between the 2‐hr cumulative CO2 excretion from trilabeled caffeine and the apparent oral metabolic clearance rate (MCR) of caffeine (r = 0.90). Monolabeled CBTs in smokers and nonsmokers demonstrated that 80% ± 4% of labeled CO2 expired in the breath during the first 2 hr of a trilabeled CBT was derived from the 3 position; at 6 to 8 hr equal amounts were derived from the 3 and 7 positions. Little N‐demethylation was observed from the 1 position at any time during the 8‐hr test. The results indicate that the 2‐hr cumulative excretion of labeled CO2 could be used to accurately predict the metabolic clearance rate of caffeine and is the best CBT parameter for detecting the effect of smoking on caffeine N‐demethylation. The data suggest that the primary routes of caffeine metabolism are 3‐N‐demethylation and ring hydroxylation and confirm that caffeine metabolites are N‐demethylated primarily in the 3 and 7 positions.

The effect of age, gender, and sexual maturation on the caffeine breath test.

This study demonstrated the feasibility of utilizing the (3-13C-methyl) caffeine breath test (CBT) in children and adolescents, and examined the effect of gender, age, and puberty on the CBT. The CBT, expressed as the 2-hour accumulative exhalation of labeled CO2 (2-hour CO2), was compared to the CBT results in the adult. The 2-hour CO2 values were higher in the children than the adult, and the decrease in the CO2 values occurred in males during late puberty and in females during early puberty.

Effects of antioxidants on metabolism of aromatic polycyclic hydrocarbons.

Abstract A number of antioxidants have been shown to inhibit the neoplastic effects of a variety of chemical carcinogens. Some of these antioxidants such as BHA and BHT are widely distributed in the environment, and it is possible that they could play a role in diminishing the impact of exposures to chemical carcinogens. The mechanism or mechanisms of these inhibitions have not been established. Two major categories seem likely: (a) some type of direct interaction between antioxidant and reactive species of carcinogen and (b) an indirect effect. Using BHA as the antioxidant and BP as the carcinogen, studies of this latter possibility have been initiated. The metabolism of BP by liver microsomes in female A/HeJ mice fed a control diet or a diet containing BHA was studied. AHH activity was not changed by BHA feeding. However, the measurement of other parameters demonstrated that BHA feeding altered the microsomal system metabolizing BP. Incubation of BP and calf thymus DNA with liver microsomes from BHA-fed mice showed about half the binding of BP metabolites to DNA as compared to that of controls. The AHH activity of mice fed BHA was much more sensitive to in vitro inhibition by α-naphthoflavone than that of controls. The amount of cytochrome P-450 was increased per unit weight of microsomal protein and liver in mice fed BHA. The ethyl isocyanide binding spectra were measured to see if alterations of cytochrome P-450 might be produced by BHA feeding. The maximum at 430 mμ was the same in control and BHA-fed mice. However, the maximum at 455 mμ was lower in BHA-fed mice than in controls, which indicated that BHA had caused some change. These data show that BHA feeding results in altered properties of liver microsomes, including a decrease in BP metabolite binding to DNA.

Effects of pregnancy on the cytochrome P-450 system in mice

The effects of pregnancy on the hepatic cytochrome P-450-dependent mixed-function monooxygenase system (P-450) from day 6 to day 18 of gestation were examined in the C57BL/6J mouse. Pregnancy induced an initial increase and then a decrease in total P-450 content, a decrease in microsomal aminopyrine-N-demethylase activity, and had no effect on microsomal ethylmorphine-N-demethylase activity. Pregnancy also induced in the C57BL/6J and the DBA/2J mice a new major isozyme of P-450 (P-450gest) as determined by high performance liquid chromatography and gel electrophoresis.

Direct measurement of aminopyrine N-demethylase and antipyrine hydroxylase activities in a monolayer rat primary isolated hepatocyte system

This paper describes a simple method for monitoring changes in aminopyrine N-demethylase and antipyrine hydroxylase activities in isolated primary hepatocyte monolayer culture. Aminopyrine N-demethylase activity was determined by monitoring the rate of formation of 14CO2 derived from the N-demethylation of [dimethylamino-14C]aminopyrine (AP). The rate of AP N-demethylation increased linearly with time for 60 min and proportionately with cell concentrations between 4.1 x 10(5) to 1.67 x 10(6) cells/incubation. As expected, non-linear AP N-demethylase kinetics were observed with hepatocytes as well as with microsomal preparations derived from control rats. Hepatocytes prepared from phenobarbital (PB)-pretreated animals exhibited increased AP N-demethylase activity and typical Michaelis-Menten kinetics. In contrast, microsomal preparations from PB-treated animals exhibited non-linear N-demethylase kinetics that differed from the kinetics of preparations derived from control animals. Antipyrine hydroxylase activity was determined by monitoring the rate of formation of non-extractable conjugated 4-hydroxyantipyrine from [N-14C-methyl]antipyrine. Antipyrine hydroxylase activity was found to increase linearly for 120 min and proportionately with cell concentrations. Antipyrine hydroxylation by hepatocytes prepared from control and PB-pretreated animals followed typical Michaelis-Menten kinetics. AP N-demethylase activity immediately after plating was 10 per cent lower than at 4 hr, whereas antipyrine hydroxylase activities were similar. Culturing hepatocytes for 24 hr resulted in a decline to 40 and 60 per cent of control for AP N-demethylase activity and antipyrine hydroxylase activity respectively.

389 CYTOCHROME P-450-P; A PREGNANCY (P) INDUCED P-450 ISOZYME IN MICE

Cytochrome P-450 mediated mixed function monooxygenases (P-450) are a group of enzymes which metabolize many substrates including steroids, environmental chemicals and drugs. P-450 in animals has been shown to be important in certain chemical induced birth defects, cancer, mutations and organ damage. Select P-450 isozymes can be induced by exposure to certain chemicals. We and others have shown that P markedly decreases maternal hepatic P-450 function. To further investigate the effect of P on P-450 we used an HPLC method (PNAS 77:6473 1980) to analyze tlhe profile of P-450 during P. Solubilized hepatic microsomes from non-pregnant female and pregnant C57BL/6 mice were compared. Comparison of elution profiles of P-450 from nonpregnant and pregnant mice show a marked increase during P in a specific peak (P-450-P) as shown. P-450-P did not co-chromatograph with the classical P-450 isozyme peaks induced by phenobarbital, 3 methylcholanthrene, or pregnenolone-16 ∝ carbonitrile. P-450-P is detectable by day 6 of gestation and remains elevated at least through post partum day 3. In summary, P induces in mice a new major P-450 isozyme peak, P-450-P, which does not co-chromatograph with the classical forms of P-450.

ETHANOL |[lpar]|E|[rpar]| METABOLISM IN PREGNANCY |[lpar]|P|[rpar]| AS DETERMINED BY THE |[lsqb]|14C|[rsqb]| ETHANOL BREATH TEST |[lpar]|EBT|[rpar]|

Maternal E consumption in P poses a known risk to the mother and fetus. The purpose of this study was to determine the effects of P on in vivo metabolism of E. The in vivo rate of E metabolism can be monitored by the EBT by measuring the rate of CO2 derived from the metabolism of radiolabeled E (Toxicol Appl Pharmacol, 61:177, 1981). The EBT was conducted in 7 pregnant (Pg) and 5 nonpregnant (Npg) C57/B6J mice of similar ages. Day 0 was when vaginal plugs were found or when Npg mice were entered into the protocol. The EBT was conducted on days 6,8,10,14, and 16. [14C] E (5×105 dpm) and unlabeled E (50 mg/kg) were administered i.p. The mice were immediately placed in air collection chambers, 14CO2 was collected by bubbling exhaled air through an alcohol trapping solution and then a CO2 trapping solution of ethanolamine methanol, and the rate of 14CO2 exhaled was determined. The elimination rate constant (Kel) of labeled CO2 was calculated and used as the measurement parameter of E clearance. The Kel did not change for Npg mice. In the Pg mice, the Kel did not differ from that of Npg mice on day 6 or 10 of gestation, but was increased on day 8 (Npg Kel=0.0592±0.002, Pg Day 8 Kel=0.0698±0.003; p<.001) and was decreased on days 14 and 16 (0.0525±0.002 and 0.0501±0.002, respectively; p<.001 vs. Npg). In summary, the rate of E metabolism in P changes biphasically. The rate of E metabolism as determined by the EBT increased significantly during organogenesis but decreased in late P.Maternal E consumption in P poses a known risk to the mother and fetus. The purpose of this study was to determine the effects of P on in vivo metabolism of E. The in vivo rate of E metabolism can be monitored by the EBT by measuring the rate of CO2 derived from the metabolism of radiolabeled E (Toxicol Appl Pharmacol, 61:177, 1981). The EBT was conducted in 7 pregnant (Pg) and 5 nonpregnant (Npg) C57/B6J mice of similar ages. Day 0 was when vaginal plugs were found or when Npg mice were entered into the protocol. The EBT was conducted on days 6,8,10,14, and 16. [14C] E (5×105 dpm) and unlabeled E (50 mg/kg) were administered i.p. The mice were immediately placed in air collection chambers, 14CO2 was collected by bubbling exhaled air through an alcohol trapping solution and then a CO2 trapping solution of ethanolamine methanol, and the rate of 14CO2 exhaled was determined. The elimination rate constant (Kel) of labeled CO2 was calculated and used as the measurement parameter of E clearance. The Kel did not change for Npg mice. In the Pg mice, the Kel did not differ from that of Npg mice on day 6 or 10 of gestation, but was increased on day 8 (Npg Kel=0.0592±0.002, Pg Day 8 Kel=0.0698±0.003; p<.001) and was decreased on days 14 and 16 (0.0525±0.002 and 0.0501±0.002, respectively; p<.001 vs. Npg). In summary, the rate of E metabolism in P changes biphasically. The rate of E metabolism as determined by the EBT increased significantly during organogenesis but decreased in late P.

THE EFFECTS OF PREGNANCY (P) ON THE RESPONSE OF THE CYTOCHROME P-450-DEPENDENT MIXED-FUNCTION MONOOXYGENASE SYSTEM (P450) TO ETHANOL EXPOSURE AND DIET CHANGE

Many xenobiotics and endogenous substrates are metabolized by P450, an enzyme system induced by certain substrates. We and others have shown that P decreases P450 in animals fed lab chow; however, little is known of how P alters the interaction of xenobiotics, diet, and P450. Pregnant (Pg) and nonpregnant (Npg) female C57BL/6J mice were maintained on lab chow and H2O ad lib before the study began. They were fed a diet containing 25% ethanol-derived calories (EDC). Controls were pair-fed a similar diet with 25% sucrose-derived calories (SDC). Day 6 of gestation was day 0 of the diet for Pg mice. The aminopyrine breath test (ABT) was done on diet days 0,2,4,8, and 10 by our previously described methods (Pediatr Res 17:150, 1983). The elimination rate constant was the ABT parameter used for comparison.The change from lab chow to SDC caused a general decrease in P450 and prevented the decrease in P450, which we have previously shown to occur in pregnancy. Exposure of Npg mice to ethanol increased P450 activity, which was not seen in the Pg animals.