Disposition of 14C-alpha-cyclodextrin in germ-free and conventional rats.

The absorption, disposition, metabolism, and excretion of uniformly (14)C-labeled alpha-cyclodextrin ((14)C-alpha-CD) was examined in four separate experiments with Wistar rats. In Experiment 1, (14)C-alpha-CD (25 microCi, 50 mg/kg bw) was administered intravenously to four male and four female conventional rats. In Experiment 2, (14)C-alpha-CD (25 microCi, 200 mg/kg bw) was given by gavage to four male and four female germ-free rats. In Experiments 3 and 4, (14)C-alpha-CD was given to groups of four male and four female conventional rats by gavage at different dose levels (100 microCi, 200 mg/kg bw; 25 microCi, 200 and 100 mg/kg bw). In all experiments, (14)C was measured in respiratory CO(2), urine, and feces over periods of 24-48 h, and in the contents of the gastrointestinal tract, blood, main organs, and residual carcass at termination of the experiments. The chemical identity of the (14)C-labeled compounds was examined by HPLC in blood (Experiment 1), urine (Experiments 1-4), feces (Experiments 2-4), and samples of intestinal contents (Experiments 2 and 4). Recovered (14)C was expressed as percentage of the administered dose. Experiment 1 showed that intravenously administered alpha-CD is excreted rapidly with urine. During the first 2h after dosing, plasma (14)C levels decreased rapidly (t(1/2), 26 and 21 min in male and female rats, respectively). About 13% of the administered (14)C dose (range 4.6-30.6) was detected in the feces, respiratory CO(2), organs, and carcass at the end of the experiment, i.e., 24 h after dosing. The presence of about 1.9% in the intestinal contents and feces suggests that a certain fraction of systemic alpha-CD is eliminated with the bile or saliva. Conclusive evidence, either positive or negative, for a hydrolysis and further metabolism of a small fraction of the administered alpha-CD by the enzymes of the mammalian body could not be gained from this experiment. Upon oral administration of (14)C-alpha-CD to germ-free rats (Experiment 2), about 1.3% of the label expired as CO(2) within 24 h. In the urine collected from 0 to 8 h after dosing, (14)C-alpha-CD was the only radiolabeled compound detected. The amounts of alpha-CD detected in the urine suggest that on average about 1% of an oral dose is absorbed in rats during small-intestinal passage. In conventional rats (Experiments 3 and 4), a delayed appearance of respiratory (14)CO(2) was observed which is attributed to the non-digestibility of alpha-CD and its subsequent microbial fermentation in the cecum and colon. In the urine collected at 4 h after dosing, a small amount of unchanged (14)C-alpha-CD was detected which confirms that about 1% of the ingested alpha-CD is absorbed intact and is excreted via the kidneys. No (14)C-alpha-CD was found in the feces. It is concluded from the data that ingested (14)C-alpha-CD is not digested in the small intestine of rats but is fermented completely by the intestinal microbiota to absorbable short-chain fatty acids. Therefore, the metabolism of alpha-CD resembles closely that of resistant starch or other fermentable dietary fibers.

Disposition of [14C]gamma-cyclodextrin in germ-free and conventional rats.

The absorption, disposition, metabolism, and excretion of 14C-labeled gamma-cyclodextrin ([14C]gamma-CD) was examined in four separate experiments with Wistar rats. In experiment 1, [14C]gamma-CD (25 microCi, 600 mg/kg body wt) was administered intravenously to four male and four female conventional rats. In experiment 2, [14C]gamma-CD (25 microCi, 1000 mg/kg body wt) was given by gavage to four male and four female germ-free rats. In experiments 3 and 4, [14C]gamma-CD (25 and 100 microCi, respectively, 1000 mg/kg body wt) was given to four male and four female conventional rats by gavage. In all experiments, 14C was measured in respiratory CO2, urine, feces, contents of the gastrointestinal tract, blood, main organs, and residual carcass. The chemical identity of the 14C-labeled compounds was examined by HPLC in urine (experiments 1-4), blood (experiments 1 and 4), and samples of intestinal contents (experiments 3 and 4). Recovered 14C was expressed as a percentage of the administered dose. Total recovery of 14C varied between about 90% (experiments 3 and 4) and 100% (experiments 1 and 2). Experiment 1 showed that about 90% of intravenously administered gamma-CD is excreted in urine within 24 h. During the first 2 h after dosing, plasma 14C levels decreased rapidly (t1/2, 15-20 min). The remaining 10% of the dose was probably excreted into the gastrointestinal tract with bile and saliva. In addition, some [14C]gamma-CD may have been degraded by plasma and tissue amylases. Since glucose is the common product of systemic hydrolysis and intestinal digestion, it is not possible to quantitate the relative flux of [14C]gamma-CD through these two pathways. Upon oral administration of [14C]gamma-CD to germ-free rats (experiment 2), about 66% of the label was expired as CO2 within 24 h. The rate of 14C exhalation reached a maximum at 90 min after dosing and then declined steadily. In the urine, and in the contents of the cecum and colon collected at 24 h, [14C]gamma-CD was not found (except for a trace in the cecum of females). In conventional rats (experiments 3 and 4), a similar, fast appearance of respiratory 14CO2 was observed. There was no delayed formation of 14CO2 due to incomplete digestion and subsequent microbial fermentation in the cecum and colon. In pooled urine collected at 4 h after dosing, a small amount of unchanged [14C]gamma-CD was detected (experiment 4). From this result, it was estimated that less than 0.02% of ingested intact gamma-CD was absorbed and excreted with the urine. It is concluded from the data that ingested [14C]gamma-CD is rapidly and essentially completely digested in the small intestine to absorbable [14C]glucose. The absorption of intact [14C]gamma-CD by passive diffusion is very low (<0.02%). Therefore, the metabolism of gamma-CD resembles closely that of starch or linear dextrins.

Metabolism and biochemical effects of 3,3',4,4'-tetrachlorobiphenyl in pregnant and fetal rats.

The metabolism and distribution of a single oral dose of 25 mumol 14C-labelled 3,3',4,4'-tetrachlorobiphenyl (14C-TCB) were investigated in pregnant female Wistar rats and their fetuses. TCB was administered on day 13 of gestation and the elimination was followed for 7 days. Non-pregnant rats were treated similarly for comparison. Fecal elimination of 14C-TCB derived radioactivity was significantly lower in pregnant rats than in non-pregnant rats. The major metabolite found in adult liver and plasma, placental tissue, whole fetuses and fetal plasma was 3,3',4',5-tetrachloro-4-biphenylol (4-OH-TCB). Tissue levels (liver, abdominal fat, skin, skeletal muscle, kidney and plasma) of 14C-TCB-derived radioactivity declined by 65-85% over a 7-day period following administration in the adult animals. However, 14C-TCB-derived radioactivity accumulated more than 100-fold in the fetuses over the same time period, and GC/MS analysis revealed that the fetal accumulation in radioactivity was due primarily to 4-OH-TCB, and not the parent compound. On day 20 of gestation, concentrations of 4-OH-TCB were 14 times greater in fetal plasma than maternal plasma. Treatment with 14C-TCB significantly reduced plasma thyroxine levels by at least 28% up to 7 days after administration in non-pregnant animals and up to 4 days after administration in pregnant rats (31% decrease). By 7 days after administration plasma thyroxine levels had returned to control levels in the TCB-treated pregnant rats. However, fetal plasma thyroxine levels were significantly decreased by 35% in fetuses from 14C-TCB-treated dams 7 days after TCB administration. Hepatic microsomal ethoxyresorufin-O-deethylase (EROD) activity was significantly induced in TCB-treated dams relative to controls at 4 and 7 days after administration, while no EROD activity was detected in hepatic microsomes from control or TCB treated fetal rats at day 20 of gestation. These data suggest that hydroxylated metabolites of polychlorinated biphenyls may play a role in the development toxicity of these compounds.

Influence of wheat bran on NMU-induced mammary tumor development, plasma estrogen levels and estrogen excretion in female rats.

In our animal experiments the hypothesis was tested that a high-fiber (HF) diet reduces tumor promotion by interruption of the enterohepatic circulation resulting in lowered estrogen exposure of the estrogen-sensitive tissue. In the first experiment the development of N-nitrosomethylurea (NMU) induced mammary tumors was investigated. One group of rats (HF) was fed a HF diet (11% fiber, based on wheat bran), the other group (LF) fed a low-fiber diet (0.5% fiber, based on white wheat flour). Tumor incidence (90 and 80%, respectively) and latency (121 and 128 days, respectively) were similar in the HF and LF groups. Compared to the LF group, HF rats had lower tumor weights (0.16 vs 0.55 g; P less than 0.01) and a slightly lower tumor multiplicity (1.8 vs 2.8 tumors per tumor-bearing rat). These differences were reduced after adjustment for body weight. In a second experiment rats, not treated with the carcinogen, were kept on the same HF and LF diets. From these rats 24-h urine and feces and orbital blood samples were collected for analysis of (un)conjugated estrogens. The excretion of both free and conjugated estrogens in fecal samples was about 3-fold higher in HF rats than in LF rats. During the basal period of the cycle urinary excretion of estrone was lower in HF rats (mean 9.7 ng/day) than in LF rats (mean 13.0 ng/day; P less than 0.05). It is concluded that wheat bran interrupts the enterohepatic circulation of estrogens, but plasma levels are not affected. Whether the development of mammary tumors is reduced by the introduction of specific components of wheat bran, or by a reduced body weight due to a lower (effective) energy intake remains to be determined.

A new radioimmunoassay for the determination of the angiotensin-converting enzyme inhibitor perindopril and its active metabolite in plasma and urine: advantages of a lysine derivative as immunogen to improve the assay specificity.

A new radioimmunoassay (RIA) was developed for the direct measurement of perindoprilate (PT), the active metabolite (diacid) of Perindopril (P), an angiotensin-converting enzyme (ACE) inhibitor. Antibodies were raised in rabbits against the lysine derivative of PT conjugated to bovine serum albumin. The p-hydroxyphenyl derivative of the lysine analogue was used for preparation of the radioligand by iodination (125I). Cross-reactivities for the glucuronide metabolites of P and PT are low (0.25 and 3.5%, respectively). The theoretical limit of detection is 0.2 nM, the sensitivity attainable with random samples is about 0.5 nM. Within- and between-assay variabilities observed were 4.2-6.7 and 2.8-5.9%, respectively (concentration range 2.1-41.7 nM). Serial dilution of plasma and urine samples showed excellent parallelism (r greater than 0.95; P less than 0.001). Recoveries of PT spiked to urine and plasma samples were 90-120%. The prodrug P can be measured in the same sample (plasma/urine) after chromatographic separation on a Dowex AG 1 x 2 anion-exchange column and quantitative alkaline hydrolysis of the P-containing fraction. It is concluded that the specificity and sensitivity of this assay are amply sufficient for pharmacokinetic studies and in patient monitoring.

In vitro synthesis of 16 alpha-hydroxyestrone by female rat liver microsomes: its possible role in the etiology of breast cancer.

Liver homogenates from female rat strains (Sprague-Dawley, Wistar and Fisher) were incubated in a NADPH regenerating medium in the presence of labelled and unlabelled estrone. Liver microsomes isolated from male rats and female mice were used as positive controls. Using HPLC and paper chromatography, under the experimental conditions used it was found that liver homogenates from female rats were able to convert estrone to various metabolites such as 16 alpha-hydroxyestrone. In a mutagenicity assay (Ames test), with 16 alpha-hydroxyesterone as test substance, two strains (TA98 and TA1538) of the five strains tested showed a 2-3-fold increase in the number of his+ revertants relative to the control values. Estrone did not cause any mutagens in the test used. It is concluded that female rats are able to synthesize 16 alpha-hydroxyestron in vitro. Whether this compound is risk factor for breast cancer remains unclear.