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The biochemical basis for the unique ability of Beagle dog liver microsomes to metabolize 2,2',4,4',5,5'-hexachlorobiphenyl (245-HCB) was investigated. The major phenobarbital (PB)-inducible cytochrome P-450 isozyme, called PBD-2, was purified to 95% homogeneity from liver microsomes of both control and PB-treated dogs, as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In a reconstituted system containing cytochrome b₅, PBD-2 metabolized 245-HCB at a rate greater than three-fold that seen in liver microsomes from PB-treated dogs. Immunoblot analysis revealed that, upon PB treatment, the increase in the level of PBD-2 in dog liver microsomes correlated well with the increase in the rate of hepatic microsomal metabolism of 245-HCB by dogs. Anti-PBD-2 IgG caused a > 90% inhibition of 245-HCB metabolism by microsomes obtained from control and PB-treated dogs. Studies were also conducted to assess the ability of PBD-2 to metabolize 2,2',3,3',6,6'-hexachlorobiphenyl (236-HCB) and 4,4'-dichlorobiphenyl (4-DCB). Dog liver microsomes readily metabolized 236-HCB, and PB treatment led to a dramatic increase in this rate of metabolism, suggesting a role for PBD-2 in the metabolism of 236-HCB. In a reconstituted system containing cytochrome b₅, PBD-2 metabolized 236-HCB at a rate greater than two-fold that observed in microsomes from PB-treated dogs. Pretreatment of microsomes obtained from PB-treated dogs with chloramphenicol (a highly selective inactivator of PBD-2) caused a nearly 70% decrease in the microsomal metabolism of 236-HCB. Anti-PBD-2 IgG inhibited by > 90% 236-HCB metabolism by microsomes from both control and PB-treated dogs. In contrast, PB treatment caused no significant alteration in the metabolism of 4-DCB by dog liver microsomes, and PBD-2 metabolized this compound poorly, even in the presence of cytochrome b₅. Taken together, these data indicate that the dog hepatic cytochrome P-450 isozyme, PBD-2, is present in microsomes obtained from both control and PB-treated animals. PBD-2 is responsible for the microsomal metabolism of 245-HCB, and this isozyme likely accounts for the unique ability of Beagle dogs to readily metabolize and eliminate this compound in vivo. The data also strongly suggest that PBD-2 is responsible for the microsomal metabolism of 236-HCB in dogs. However, PBD-2 is not likely involved in the microsomal metabolism of 4-DCB by this species.

The tail-cast suspension system was used as a ground-based model of weightlessness to study the effects of reduced weight-bearing (unloading) and attendant atrophy on carbohydrate metabolism in the rat soleus muscle. Changes in glycogen metabolism during unloading were biphasic. The initial phase, which took place during the first 24 hours, was characterized by increased glycogen concentrations, the result of decreased glycogenolysis. This glycogen accumulation in turn decreased the glycogen synthase activity ratio. These alterations were maintained thereafter. A similar glycogen increase was seen in the soleus unloaded by hypogravity. A rapid decline in glucose uptake also occurred during this initial phase of unloading, both in the absence and presence of insulin. During the second phase of this unloading response, beyond 24 hours, there was an increase in the sensitivity of the soleus to insulin for glucose uptake that coincided with the onset of muscle atrophy. However, this increased insulin sensitivity was not seen for glycogen synthesis, indicating differential regulation of these two processes by insulin. The enhanced insulin sensitivity likely resulted from an increase in the binding capacity for the hormone, resulting from no increased turnover of the insulin receptor during unloading. Additionally, the onset of increased insulin sensitivity coincided with a return to normal of basal glucose uptake, despite the continued unloading. When the 3-day unloaded soleus was reloaded, carbohydrate metabolism returned to normal after a triphasic response. Within two hours, the glycogen concentration decreased below control levels, due to increased glycogenolysis. During the second phase of reloading, from two to 24 hours, glycogen accumulated dramatically due to an enhanced capacity of the muscle for glycogenesis. Insulin sensitivity returned to normal by 24 hours of reloading. In the final phase, beyond 24 hours, glycogen decreased to control values. An uncoupling of the inverse relationship between glycogen and the activity ratio of glycogen synthase was seen beyond four hours of reloading. These effects of unloading and reloading were not due to systemic factors, as the extensor digitorum longus was unaffected. It is apparent that the unloaded soleus is a potent model with which to study the individual factors that regulate carbohydrate metabolism in skeletal muscle.

The suckling rat obtains about 70% of its energy from the catabolism of lipid, compared to only about 25% in the adult rat. In addition, lipids are in great demand for membrane synthesis in the rapidly growing tissues. Thus the suckling rat represents an important system in which to study lipid metabolism. In this dissertation research the following experiments were performed on rats during the suckling period: (1) Determination of the concentration and fatty acid composition of triacylglycerols, cholesteryl esters, phospholipids, and free fatty acids in dam's milk, and in suckling rat lymph, portal plasma, vena cava plasma and liver; (2) Determination of positional distribution of fatty acids among the three different positions in triacylglycerols of milk, lymph, plasma and liver; (3) Isolation of plasma and mesenteric lymph lipoproteins by density gradient centrifugation, and identification of the individual classes of lipoproteins by measurement of their density and by polyacrylamide gel electrophoresis; (4) Determination of the concentration of each lipoprotein by refractometry and determination of the concentration and fatty acid composition of different lipid components in each lipoprotein fraction; (5) Studies of physical properties of the lipoproteins using analytical ultracentrifugation; (6) Determination of apoprotein composition and concentration in each lipoprotein fraction. The results show that the suckling rat consumes a diet which is rich in medium chain fatty acids (35% of total milk fatty acids). Mesenteric lymph triacylglycerols carry a significant amount of these medium chain fatty acids, unlike the adult in which they would be transported as free fatty acids, unlike the adult in which they would be transported as free fatty acids via the portal vein. Medium chain fatty acids are enriched at the sn-3 position in all triacylglycerols. Lymph contains chylomicrons, very low density lipoproteins, and high density lipoproteins, but no low density lipoproteins. Plasma contains all classes of lipoproteins but at concentrations higher than found in the adult, especially low density lipoproteins which are present at levels six times that found in the adult. The plasma very low density lipoproteins have a lipid composition which resembles that of chylomicron remnants. Apoprotein, lipid composition, and electrophoresis patterns show that the plasma lipoproteins in the suckling rat are heterogeneous.

To determine the structure(s) of the spermidine conjugate excreted into urine, extensive pharmacokinetic studies on the turnover of [¹⁴C]spermidine were conducted in both rats and humans. These studies demonstrated that exogenously administered [¹⁴C]spermidine equilibrated with endogenous polyamine pools. Radiolabeled urine collected from the humans was subjected to a cleanup protocol and subsequent analysis by GC-MS, which demonstrated the presence of N-acetylspermidine. Further analysis of the radiolabeled urine by thin layer chromatography (TLC) demonstrated the presence of both N¹- and N⁸-acetylspermidine in an approximate 1 to 1 ratio. Using similar methodologies the monoacetyl derivatives of putrescine and cadaverine were found to be the primary conjugated products of these polyamines excreted into human urine. Radiolabeled rat tissue extracts, analyzed by TLC, demonstrated that all tissues studied contained [¹⁴C] N¹- and N⁸-acetylspermidine. N¹-acetylspermidine was the primary isomer detected in all tissues; although, N⁸-acetylspermidine was detected in all tissues studied. The N-acetylspermidine content of an isolated cell system was determined in Chinese hamster ovary cells. These cells contained both N¹- and N⁸-acetylspermidine in an approximate 2 to 1 ratio. To directly measure the N-acetylpolyamines excreted into urine, two high performance liquid chromatography (HPLC) methods were developed. Both HPLC methods utilize a cation exchange resin, one using high pH, low salt buffers and the other low pH and high salt buffers. The primary N-acetylpolyamine excreted into human urine is N-acetylputrescine with lesser amounts of N¹- and N⁸-acetylspermidine which exist in a 1 to 1 ratio. In contrast, cancer patients excreted elevated amounts of both N-acetylputrescine and N¹-acetylspermidine. Cystic fibrosis patients were also found to excrete elevated amounts of N¹-acetylspermidine resulting in a consistently elevated N¹- to N⁸-acetylspermidine ratio. Mice injected with P-388 leukemia tumor excreted elevated amounts of N-acetylputrescine, N¹-acetylspermidine and N⁸-acetylspermidine. In contrast, the excretion of the unconjugated polyamines putrescine and spermidine in these animals was decreased, suggesting that the altered polyamine excretion was not primarily due to the presence of the tumor. Administration of Adriamycin to the tumor bearing animals resulted in the elevation of N¹-acetylspermidine excretion which was proportional to the relative tumor burden. Similar results were obtained from 2 human leukemia patients studied following chemotherapy.

Phencyclidine (PCP) has become a major drug of abuse in recent years, and treatment of PCP overdose thus becomes a major concern. This dissertation was designed to examine the pharmacokinetics of PCP in dogs and to provide a rational basis for overdose treatment. One method of treating toxicity was also tested. Before any animal studies could begin, however, analytical procedures for PCP and its metabolites had to be developed. Three gas chromatographic procedures capable of analyzing PCP, two monohydroxymetabolites (PCHP and PPC), and a pentanoic acid metabolite (PCAPA) were developed. Each assay was validated for accuracy and precision. The analytical methods for the metabolites of PCP were up to 50 times more sensitive than others previously reported in the literature. The analytical method for PCP, PCHP and PPC was also able to separate the cis and trans forms of PPC. Animal studies were performed to determine the pharmacokinetic behavior of PCP in dogs. From these experiments, PCP was determined to have a very high clearance (Cls) value approaching the upper limits of hepatic blood flow. PCP has a very large volume of distribution (V) and relatively short half-life (t(, 1/2)). The clearance of PCP was mainly due to metabolism and not to excretion of the unchanged drug. Radioactivity studies showed total recovery ranging between 65 and 80% of the dose administered. PCP was primarily metabolized to PCAPA, PCHP, and PPC. PCHP and PPC were very quickly conjugated. Bioavailability ranged between 15 and 50%. Oral clearance, an estimate of intrinsic clearance, was very large. Pharmacokinetic parameters of PPC and PCHP were similar to each other and comparable to PCP. The administration of activated charcoal increased the clearance of PCP but this increase was not statistically significant from controls.

Part I. The disposition kinetics of 5-fluorouracil (5-FU) and a 5-FU prodrug, 5'-deoxy-5-fluorouridine (5'dFUR), were investigated in twelve patients with colorectal carcinoma. Each patient randomly received either two single, intravenous (iv) doses of 5-FU (7.5 and 15 mg/kg) or 5'dFUR (2 and 4 g/m²) on separate days. In addition, the whole blood/plasma concentration ratio and stability of 5-FU and 5'dFUR at 37°C was determined in whole blood from normal volunteers and in five patients with colorectal carcinoma. A disproportionate increase in area under the curve and corresponding decrease in total body clearance with increasing dose was observed suggesting dose-dependent behavior of both 5-FU and 5'dFUR. The renal clearance of both compounds was independent of dose. Therefore, the mechanism for dose-dependence appears to be primarily due to nonlinear elimination associated with nonrenal processes. The mean 5-FU half-life following the high dose was nearly twice as long as that observed for the low dose suggesting product-inhibition as a possible explanation for the observed nonlinearity in 5-FU elimination. The present studies demonstrate that both 5-FU and 5'dFUR degrade when incubated in whole blood. However, the estimated whole blood clearance does not contribute significantly to the observed total body clearance values. Part II. This investigation examined the influence of gender and cigarette smoking on the disposition of propranolol (P) and metoprolol (M) after both oral and iv administration to twenty healthy volunteers. The only significant differences between smokers and nonsmokers were a higher total body clearance of P and a larger volume of distribution of M in smokers. A comparison of oral clearance values demonstrated that smokers and nonsmokers had a similar capacity to metabolize P and M. Differences between males and females were not observed for any of the pharmacokinetic parameters examined. Nonlinearity in the absorption of P and M was observed and the data suggest marked intersubject differences in the ability of the liver to extract or metabolize these drugs on their first-pass through the liver.

No description available.